WO2004085644A2 - Methode de production d'organismes recombinants - Google Patents

Methode de production d'organismes recombinants Download PDF

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Publication number
WO2004085644A2
WO2004085644A2 PCT/EP2004/002881 EP2004002881W WO2004085644A2 WO 2004085644 A2 WO2004085644 A2 WO 2004085644A2 EP 2004002881 W EP2004002881 W EP 2004002881W WO 2004085644 A2 WO2004085644 A2 WO 2004085644A2
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nucleic acid
acid sequence
sequence
dna
plant
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PCT/EP2004/002881
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WO2004085644A3 (fr
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Ralf Badur
Bernd Reiss
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Basf Plant Science Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination

Definitions

  • the present invention relates to methods for producing a recombinant eukaryotic organisms, preferably a recombinant plant, comprising the step of decreasing the chromatin assembly capacity of said organism.
  • the method may be used for introducing sequences into the chromosomal DNA of a eukaryotic cell either by illegitimate insertion or, preferably, by homologous recombination.
  • the method results in increased transformation efficacy and/or increased frequency of homologous recombination.
  • the invention furthermore relates to expression constructs suitable for expressing at least part of a nucleic acid sequence encoding for a chromatin assembly factor protein (e.g., in form of antisense or double-stranded RNA) suitable for decreasing expres- sion or functionality of at least one chromatin assembly factor protein in plant or plant cells.
  • a chromatin assembly factor protein e.g., in form of antisense or double-stranded RNA
  • Object of the invention are furthermore recombinant plant cells or plants comprising a decreased chromatin assembly capacity, preferably a decreased expression or functionality at least one chromatin assembly factor and the use of said recombinant plants for the production of food, feeds, seed, pharmaceuticals or fine chemicals.
  • Transformation is a basic tool in the generation of recombinant ' organisms.
  • a variety of species especially plant species
  • transformation is a relatively labor and time consuming process. Therefore, any improvement of this process leads to substantial improvements in the generation of recombinant organisms.
  • the production of correspondingly modified eukaryotic organisms can be realized only for .
  • Transgenic animal models for human diseases have lead to remarkable breakthroughs, revealing the molecular basis of numerous illnesses . These discoveries are already influencing disease diagnosis, treatment and even cures. Numerous transgenic animals have been created in the development of transgenic technology. Transgenic animals have provided models for human diseases. While most of these investigations have been performed using transgenic mice, studies are now emerging on other trans.genic animals, demonstrating a wealth of biomedical, pharmaceutical, and agricultural implications (US 6,147,202). Notwithstanding the technologies now available for creating rodent models for various diseases, these models are not always appropriate in studying human disorders. Extending transgenesis approaches to other animals (like e.g., nonhuan primates) would further enhance the utility of these models .
  • the transfer of genes into a given cell is also at the root of gene therapy.
  • one of the problems is to succeed in causing a sufficient quantity of. nucleic acid to penetrate into cells of the host to be treated and "to have said nucleic acid integrated into the host genome, especially when a directed integration is required (e.g., in case of a gene repair approach).
  • One of the approaches selected in this regard has been the integration • of the nucleic acid into viral vectors , in particular into retroviruses , adenoviruses or adeno-associated viruses.
  • US 5,928,638 describes a method of enhancing nucleic acid integration during gene transfer and gene therapy by increasing the proportion of hematopoetic stem cells in active cell cycle prior to transduction by contacting said stem cells with hydroxyurea. Hydroxyurea is known to cause direct DNA damage which is potentially linked to the risk of unwanted mutations.
  • WO 01/73001 is describing a method for genetically modifying a cell comprising the step of providing cells with synchronized cell cycle. The method is preferably carried out by utilizing triplex-forming oligonucleotides.
  • sequence-unspecific in- duction of DNA strand breaks is disadvantageous because of the potential mutagenic effect. Sequence-specific induction of DNA strand-breaks may also increase efficiency of HR but is, limited to artificial scenarios (Siebert R, Puchta H (2002) Plant Cell 14(5) :1121-31) .
  • DNA in eukaryotic cells is tightly packaged into chromatin.
  • the smallest sub-units of chromatin are the nucleosomes which consist of histones.
  • the nucleosomes form not well defined higher order structures of chromatin.
  • the tight chromatin structure has to loosen up for replication in the S-phase and the nucleosomes have to be re-assembled on DNA by successively recruiting histones to the replication fork after replication. Recruitment of histones and reconstitution of chromatin is an ordered process and requires the activity of several genes .
  • the assembly of DNA into chromatin involves a range of events, beginning with the formation of the basic unit, the nucleosome, and ultimately giving rise to a complex organization of specific domains within the nucleus.
  • the first step is the deposition onto the DNA of a tetramer of newly synthesized (H3-H4)2 to form a sub-nucleosomal particle, which is followed by the addition of two H2A-H2B dimers.
  • This produces a nucleosomal core particle consisting of 146 base pairs of DNA wound around the histone octamer.
  • This core particle and the linker DNA together form the nucleosome.
  • chromatin assembly is facilitated by several factors. One of them is chromatin assembly factor 1 (CAFl).
  • CAFl consists of three subunits, pl50, p60 and p48, originally " identified biochemically in human cells.
  • CAFl in yeast is dispensable, but mutations impair transcriptional silencing and si- lencing at mating type loci suggesting that CAFl plays a role in the maintenance of chromatin (Ridgway P et al. (2000) J Cell Sci 113:2647-2658; Almouzni G et al . (2OO0 " Chapter 2 : Chromatin assembly DNA replication and repair. In "Chromatin and gene expression", Frontiers in Molecular Biology, Eds. : J. Workman, S. Elgin, Oxford University Press, 2nd Edition, pp. 24-48; Ridgway P. et al. (2001) J Cell Sci 114:2711-2712).
  • CAF-1 is believed to assemble histone octamers onto replicating DNA (Kaufman PD et ⁇ -* al. (1995) Cell 81: 1105-1114; Verreault A et al . (1996) Cell 87:95-104).
  • CAFl homologous are found in a variety of eukaryotic organisms, including human, mouse, worms, fish and several plant species.
  • the nucleic acid sequence of CAF (FAS1) from Arabidop- sis thaliana is described (GeneBank Ace ' . No. BAA77811) and encodes a 815 amino acid protein.
  • a similarity to the pl50 subunit of human chromatin assembly factor-l (hCAF-I) is annotated.
  • the Arabidopsis CAF (FAS1) mutant was shown to be defective in a plant homologue of the pl50 subunit of CAFl (Kaya H et al . (2000) Plant Cell Physiol 41:1055-1066; Kaya H et al . (2001) Cell 104:131-142).
  • a CAFl function in plants in cellular organization of apical meristems and meristem- restricted gene expression was suggested.
  • a first embodiment of the invention relates to a method for modifying a chromosomal DNA-sequence in a eukaryotic cell, comprising : a) providing one or more eukaryotic cells comprising a decreased chromatin assembly capacity, wherein said eukaryotic cells is derived from or able to form a multicellular organism, and
  • said eukaryotic cells is derived from or able to form a multicellular organism. More preferred said eukaryotic cell is derived from or able to form a higher plant or mammalian organism. Most preferred said eukaryotic cell is derived from or able to form a vascular plant.
  • the modification of the chromosomal DNA sequence may be selected from the group consisting of deletion, insertion, substitution, strand break, and adduct formation.
  • the modification may be mediated by contacting said cell with a DNA-modifying molecule, which may be selected from (but shall not be limited to) the group consisting of double-stranded DNA molecules, single- stranded DNA molecules, triplex forming oligonucleotides, double-stranded RNA molecules, single-stranded RNA molecules, and proteins with nuclease or recombinase activity.
  • the method results in increased transformation efficacy (e.g., in case of illegitimate integration) and/or increased frequency of homologous recombination (in case of e.g., integration by homologous recombination) .
  • the modification of the chromosomal DNA sequence may be mediated by at least one method selected from the group consisting of
  • the decrease in chromatin assembly capacity can be mediated in numerous ways, including but not limited to decreasing expression or functionality of at least one chromatin assembly factor.
  • the chromatin assembly factor may be - for example - selected from the group consisting of CAF-1 pl50, CAF-1 p60, CAF-1 p48, PCNA, Asfl, Hatl, Asfl, Hirl, Hir2 , Hir3 , Hpc2 , and histone deacetylases.
  • Various methods are known in the art to decrease expression or functionality of a chromatin assembly factor, preferably including but not limited to
  • RNA a double-stranded RNA of a chromatin assem- bly factor encoding nucleic acid sequence (CAF dsRNA) or an expression cassette (s) ensuring the expression thereof,
  • CAF dsRNA chromatin assem- bly factor encoding nucleic acid sequence
  • the method of the invention may include the step of inducing an homologous recombination (HR) .
  • the HR may take place between a pair of DNA homology-sequences A and A' having a sufficient length and homology between each other to allow homologous recombination among A and A' .
  • HR between A and A' may constitute an intramolecular or an intermolecular recombination event .
  • the two homology-sequences A and A' undergoing homologous recombination may - for example - be both localized on one strand of a chromosomal DNA-sequence, e.g., in form of direct repeats.
  • HR will cause dele- tion of the sequences localized between A and A' .
  • This may be utilized, e.g., for deleting selection marker sequences from the chromosomal DNA.
  • two homology- sequences e.g., A and A'
  • a and A' are localized on separate DNA molecules.
  • one of the two homology sequences A and A' undergoing homologous recombination is localized on a chromoso- mal DNA-sequence.
  • the other sequence may - for example - be localized on a DNA-construct, which may function as a gene targeting construct.
  • mutations are introduced into the chromosomal DNA as a consequence of the HR between A and A' .
  • the method of the invention comprises the steps of
  • DNA-construct comprises at least one homology- sequence A having a sufficient length and homology to at least one part A' of said chromosomal DNA sequence to facilitate homologous recombination among A and A' , and wherein said DNA-construct introduces said mutation into said chromosomal DNA-sequence in consequence of the homologous recombination between A and A' , and
  • the DNA-construct comprises two homol- ogy-sequences A and B having a sufficient length and homology to at least a part A' and B' of said chromosomal sequence, respectively, to allow homologous recombination between A and A' , and B and B', respectively.
  • a and B additional sequences might be localized (like e.g., expression cassettes, functional elements) which by the homologous recombination are introduced into the chromosomal DNA.
  • the mu- tation may - for example - be introduced directly by replacement of A by A' , wherein A differs from A' by the mutation to be introduced.
  • the mutation may - for example - be introduced by insertion of the sequences comprised between A and B into the chromosomal DNA.
  • the mutation may comprise modification (e.g., base change) of the sequence localized between A' and B' .
  • Another embodiment of the invention relates to a method of producing a recombinant, eukaryotic cell by modifying a chromosomal DNA-sequence of said eukaryotic cell by a method of the invention.
  • said eukaryotic cells is derived from or able to form a multicellular organism, preferably a vascular plant .
  • Another embodiment of the invention relates to a method of producing a recombinant, eukaryotic organism, preferably a vascular plant, comprising the steps of
  • the method for modifying a chromosomal DNA sequence, or producing a recombinant eukaryotic cell or organism may in a preferred embodiment further comprises the steps of
  • the method of the invention for producing a recombinant organism further comprises the step of
  • an organism e.g., a plant ( with normal chromatin assembly capacity comprising said modification of the chromosomal DNA-sequence (e.g., the mutation introduced by the DNA-construct) .
  • the segregation may be carried out by any method known in the art (e.g., crossing and selection), whereby selec- tion may be easily realized by using standard techniques (e.g., PCR or marker technology) to monitor segregation of the mutation.
  • Another embodiment of the invention relates to novel polypeptide sequences encoding chromatin assembly factors isolated e.g., from Brassica napus, Oryza sativa and Glycine max. Another embodiment of the invention relates to isolated nucleic acid sequence encoding said polypeptide sequences of the invention.
  • dsRNA double-stranded RNA
  • Said dsRNA molecules may comprise,
  • RNA sequence which is essentially identi- cal to at least part of a nucleic acid sequence coding for a chromatin assembly factor
  • RNA sequence which is essentially complementary to at least part of said first RNA sequence under i) .
  • Another embodiment of the invention relates to recombinant expression cassettes comprising at least part of a nucleic acid sequence coding for a chromatin assembly factor under control of a promoter sequence functional in eukaryotic cells, wherein said sequence in ocalized in antisense-orientation with reagrd to said promoter.
  • a recombinant expression cassettes comprising a nucleic acid sequence of the invention encoding a novel chromatin assembly factor, or a nucleic acid sequence encoding a double-stranded RNA molecule of the invention.
  • sequences are in operable linkage with a promoter functional in plant cells.
  • the recombinant expression cassette of the invention may lead to expression of sense-, antisense or double-stranded RNA of at least part of a nucleic acid sequence coding for chromatin assembly factor.
  • Another embodiment of the invention is related to recombinant expression vectors comprising at least one nucleic acid sequence of the invention encoding a novel chromatin assembly factor or a recombinant expression cassette of the invention.
  • Another embodiment of the invention relates to recombinant organisms comprising at least one recombinant expression vector or a recombinant expression cassette of the invention.
  • the recombinant organisms is a vascular plant.
  • Another embodiment of the invention is related to a recombinant plant organism having a decreased expression or functionality of at least one chromatin assembly factor.
  • said decreased expression or functionality is caused by transformation of said plant with at least one recombinant expression vector or recombinant expression cassette of the invention or my mutating at least one endogenous gene coding for a chromatin assembly " " factor.
  • Another embodiment of the invention relates to use of at least one isolated nucleic acid sequence of the invention encoding, a novel chromatin assembly factor, at least one double-stranded RNA molecule of the invention, at least one recombinant expression cassette or recombinant expression vectors of the -invention in a method for introducing a mutation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell.
  • Another embodiment of the invention relates to a method for facilitating plant breeding utilizing a plant organism having a decreased activity or expression of at least one a chromatin assembly factor.
  • said plant organism is a plant of the invention.
  • nucleic acid refers to deoxyribonucleotides or ribo- nucleotides and polymers or hybrids thereof in either single-or double-stranded, sense or antisense form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which 7 - are me- tabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phos- phorothioates , phosphoramidates , methyl phosphonates , chiral- methyl phosphonates, 2-0-methyl ribonucleotides , peptide-nucleic acids (PNAs), and the like.
  • nucleic acid is used interchangeably herein with
  • nucleic acid sequence refers to a ' consecutive list of abbreviations, letters, characters or words, which represent nucleotides.
  • a nucleic acid can be a "probe” which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length.
  • a "target region" of a nucleic acid is a por- tion of a nucleic acid that is identified to be of interest.
  • a "coding region" of a nucleic acid is the portion of the nucleic acid which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences.
  • the coding region is said to encode such a polypeptide or protein.
  • antisense is understood to mean a nucleic acid having a sequence complementary to a target sequence, for example an mRNA sequence the blocking of whose expression is sought by hybridization with the target sequen ⁇ e.
  • the term "sense” is understood to mean a nucleic acid having a sequence which is homologous or identical to a target sequence, for example a sequence which binds to a protein transcription factor and which is involved in the expression of a given geneT
  • the nucleic acid comprises a gene of interest and elements allowing the expression of the said gene of interest.
  • gene refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the polypeptide in some manner.
  • a gene includes- untrans- lated regulatory regions of DNA (e. g. , promoters, enhancers, repressors, etc.) preceding (upstream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons).
  • coding region when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule.
  • the coding region is bounded, in eukaryotes, on the 5 ' side by the nucleotide triplet "ATG” which encodes the initiator methionine and on the 3 ' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA) .
  • ATG nucleotide triplet
  • genomic forms of a gene may also include sequences located on both the 5 ' and 3 ' end of the sequences which are present on the RNA transcript .
  • flanking sequences or regions are referred to as “flanking" sequences or regions (these flanking sequences are located 5 'or 3 ' to the non- translated sequences present on the mRNA transcript) .
  • the 5 ' flanking region may contain regulatory sequences such as pro- moters and enhancers which control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences which direct the termination of transcription, posttranscrip- tional cleavage and polyadenylation.
  • polypeptide refers to a polymer or oligomer of consecutive amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • polypeptide refers to amino acids joined .to each other by peptide bonds or modified peptide bonds and may contain modified amino acids other than the 20 gene- encoded amino acids.
  • the polypeptides can be modified by either natural processes, such as post-translational processing'; or by chemical modification techniques which are well known in the ' " -* art.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino* acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code,- as well as those amino ac.ids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine .
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e. g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium) .
  • amino acid sequence refers to a list of abbreviations, letters, characters or words representing amino acid residues.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one- letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides likewise, may be referred to by their commonly accepted single-letter codes.
  • A alanine
  • B asparagine or aspartic acid
  • C cysteine
  • D aspartic acid
  • E glutamate
  • F phenylalanine
  • G glycine
  • I isoleucine
  • K lysine
  • L leucine
  • M methionine
  • N asparagine
  • P proline
  • Q glutamine
  • R arginine
  • S serine
  • T threonine
  • V valine
  • W tryptophan
  • Y tyro- sine
  • Z glutamine or glutamic acid
  • isolated means that a material has been removed from its original environment.
  • a natu. rally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated " .
  • Such polynucleotides can be ' part of a vector and/or such polyn ⁇ cie " btides or polypeptides could be part of a composition, and would be isolated in that such a vector or composition is not part of its original envi- ron ent .
  • wild-type means " “* with respect to an organism,' polypeptide, or nucleic acid sequence, that said organism is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.
  • nucleic acid sequence or a organism, expression cassette or vector com- prising said nucleic acid sequence
  • recombinant refers to all those constructs originating by recombinant methods in which either
  • nucleic acid sequence a) for example a promoter, or
  • Natural genetic environment refers to the natural chromosomal locus in the organism of ori- gin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, very especially preferably at least 5000 bp, in length.
  • the term "recombinant" with respect to nucleic acids as used herein means that the nucleic acid is covalently joined and adjacent to a nucleic acid to which it is not adjacent in its natural environment.
  • "Recombinant" polypeptides or proteins refer to polypeptides or ' proteins produced by recombinant DNA 7 techniques, i. e. , produced from cells transformed by an exogenous recombinant DNA construct encoding the desired polypeptide or protein.
  • Recombinant nucleic acids and polypeptide may also comprise molecules which as such does not exist in nature but are modi- ' ⁇ fied, changed, mutated or otherwise manipulated by man.
  • a "recombinant polypeptide” is a non-naturally occurring polypeptide that differs in sequence from a naturally occurring polypeptide by at least one amino acid residue.
  • Preferred methods for producing said recombinant polypeptide and/or nucleic acid may comprise directed or non-directed mutagenesis, DNA shuffling or other methods of recursive recombination, an especially pre- ferred method to obtain such recombinant molecules may involve gene shuffling. Shuffling methods are known in the art and, for example, described in WO 01/12817 and in the references cited therein, employed.
  • heterologous nucleic acid sequence or “heterologous DNA” are used interchangeably to refer to a nucleotide sequence which is ligated to a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
  • Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed.
  • Synthetic polypeptides or proteins are those prepared by chemical synthesis (e. . g., solid-phase peptide synthesis). Chemical peptide synthesis is well known in the art (see, e. g. , Merrifield (1963), Am. Chem. Soc . 85: 2149-2154; Geysen et al . (1984) Proc Natl Acad Sci USA 81:3998) and synthesis kits and automated peptide synthesizer are commercially available (e.g., Cambridge Research Biochemicals , Cleveland, United Kingdom; Model 431A synthesizer from Applied Biosystems, Inc., Foster City, CA) . Such equipment provides ready access to the peptides ' of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques . u ,• «
  • identity as used herein with respect to two nucleic acid sequences is understood as meaning the identity calculated with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG) , Madison, USA; Altschul et a 7 (1997) Nucleic Acids Res. 25:3389 et seq. ) , setting the following parameters:
  • Gap weight 50 Length weight: 3
  • sequence which has at least 60% homology with sequence SEQ ID NO: 1 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above program algorithm with the above parameter set, has at least 60% identity.
  • the scoring matrix blosum62 was used.
  • identity as used herein with respect to two polypeptides is understood as meaning the identity calculated with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:
  • Gap weight 8 Length weight : 2
  • sequence which has at least 60% homology with se- quence SEQ ID NO: 2 at the protein level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 2 by the above program algorithm with the above parameter set, has at least 60% identity.
  • homology has the same meaning as “identity” in the context of nucleotide sequences. However, with respect to amino acid sequences, “homology” includes the percentage of identical and conservative amino acid substitutions . Percentages of homology can be calculated according to the algorithms of Smith and Waterman (1981) Adv Appl Math 2:482 and Needle an & Wunsch (1970) J Mol Biol 48:443-453 using the scoring matrix blosum62.
  • two sequences are "substantially identical" when they have at least 99.5% nucleotide identity, when compared and a- ,• « . capitad for maximum correspondence, as measured using the known sequence comparison algorithms described above.
  • synonymous codons in a coding region may be treated as as identical to account for the degeneracy of the genetic code. '
  • the region for determination of substantial identity must span at least about 20 residues, and most commonly the sequences are substantially identical over at least about 25-200 residues .
  • two sequences are “substantially identical” when they have at least 99.5% identity, when compared and aligned for maximum correspondence, as measured using the known sequence comparison algorithms described above.
  • conservative amino acid substitutions may be treated as identical if the polypeptide substantially retains its biological function.
  • Hybridization refers to the process by which a nucleic acid strand joins with a complementary strand through hydrogen bonding at complementary bases.
  • Hybridization assays can be sensi- tive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations.
  • Stringent conditions are defined by concentrations of salt or formamide in the pre-hybridization and hybridization solutions, and/or by the hybridization temperature, and are well known in the art. Stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • the hybridization conditions for DNA:DNA hybrids preferably comprise 0.1 x SSC and temperatures between about 20°C and 45°C, preferably between about 30°C and 45°C.
  • the hybridization conditions for DNA:RNA hybrids preferably comprise 0.1 x SSC and temperatures between about 30°C and 55°C, preferably between about 45°C and 55°C. These temperatures stated for the hybridization are melting temperatures calculated by way of example for a nucleic acid with a length of about 100 nucleotides and a G + C content of 50% in the absence of tréforma-,- « mide.
  • stringent hybridization conditions include 42°C in 50% formamide ⁇ 5X SSPE, 0.3% SDS, and 200 ng./ml sheared and denatured salmon sperm DNA, and equivalents thereof. Variations on the above ranges and conditions are well known in the art . -
  • variant refers to polynucleotides or polypeptides of the invention modified at one or more nucleotides or amino acid residues (respectively) and wherein the encoded polypeptide or polypeptide retains its natural activity.
  • Variants can be produced by any number of means including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagene- sis, gene reassembly, gene site-saturated mutagenesis or any combination thereof.
  • Constantly modified variants applies to both amino acid' and nucleic acid sequences .
  • conservatively modified variants refer bo those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • degenerate codon substitutions may be achieved by ge- nerating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-bases and/or deoxyinosine residues (Batzer et al . (1991) Nucleic Acid Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260 :2605-2608; .
  • Every nucleic acid sequence recited herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except- AUG, which, along with GUG in some organisms, is ordinarily the only codon for methionine, and TGG, which is ordinarily the 4 only codon for tryptophan
  • TGG which is ordinarily the 4 only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic a- cid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, in- terspecies homologs, and alleles of the invention.
  • the term "cell” refers to a single cell.
  • the term “cells” refers to a population of cells.
  • the population may be a pure population comprising one cell type. Likewise, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise.
  • the cells may be synchronize or not synchronized, preferably the cells are synchronized.
  • chromatin assembly factor such as the CAF-1 pl50 protein (e.g., BAA77811) are considered key elements incorporating heterologous DNA into the chromosomal DNA of a plant. Transformation efficacy per se is increased by causing a decrease in chromatin assembly capacity. Furthermore, the efficacy of homologous recombination in plants is significantly increased and outperforms the methods described , in the art. The method of the invention can be realized without expressing a (foreign) protein. This has considerable advantages for approval and acceptance by the consumer.
  • the term "chromatin” as used herein is intended to mean the structure composed of DNA and proteins" and localized in the cellular nucleus presenting the carrier of genetic information. The fundamental unit of chromatin, termed the nucleosome, is com- posed of DNA and histone proteins.
  • chromatin assembly is intended to mef ⁇ i the assembly of DNA, preferably newly replicated DNA, into nucleosomes (prefer is de novo nucleosome assembly) .
  • This process includes several steps known in the art, which are - individually and in combination - summarized under the term chromatin assembly.
  • De novo nucleosome assembly occurs through a stepwise mechanism whereby histones H3 and H4, which contact the central portion of the nucleosomal DNA, are deposited first.
  • Hi-stones H2A and H2B which contact the DNA near the ends of the nucleosome core, are added subsequently.
  • HATs B- type histone acetyltransferases
  • H3 and H4 form stable complexes with a number of assembly factors that escort histones to the replication fork and facilitate their regulated deposition onto DNA.
  • HDACs histone deacetylases
  • chromatin assembly means the process of binding/deposition of histones H3 ⁇ and H4 to DNA.
  • chromatin assembly means the assembly of chromatin by binding of newly synthesized histones H3 and H4 to newly replicated DNA. ...
  • chromatin assembly capacity as used herein is preferably intended to mean the time necessary for a cell to assem- ble newly replicated DNA into chromatin.
  • a “decrease in chroma- tin assembly capacity” is intended “ to mean a prolongation of the time necessary to assemble newly replicated DNA into chromatin, preferably caused or mediated by one of the methods exemplified herein, in comparison to a cell to which said method was not ap- plied under otherwise unchanged conditions .
  • Chromatin assembly can be monitored by a variety of procedures ' " including, for example, microscopic observation, time lapse videomicroscopy (Ladoux B et al. (2000) Fast kinetics of chroma- tin assembly revealed by single-molecule video-microscopy and scanning force microscopy. Proc Natl Acad Sci USA 97:14251-', 14256), observation that the assembly of nucleosomes introduces negative superhelical turns into the DNA . (supercoiling assay) or an assay based on the differential sensitivity to micrococcal nuclease of nucleosomes and linker DNA (micrococcal nuclease assay) (Menut S. et al. (1999) Advances in Molecular Biology: A comparative Methods Approach to the Study of Oocytes and Embryos, pp.196-226, Oxford University Press, Ed J.D. Richter) .
  • a decrease in chromatin assembly capacity can also be monitored indirectly by monitoring cell cycle.
  • Decrease in chromatin assembly capacity blocks or delays entry into and progression of cells through S-phase.
  • Cells with decreased chromatin assembly capacity experience a delay in cell cycle maximal- sion after DNA synthesis but prior to anaphase (G2/M delay) .
  • Said delay may be in the range of at least 10, preferably 30 to 45 minutes .
  • chromatin assembly factor (hereinafter also CAF or CAF protein) as used herein in intended to include all cellular components which enable or facilitate the formation of nucleosome cores without being part of the final reaction product.
  • histone-interacting factors e.g., acidic factors which can form complexes with histones and enhance the process of histone deposition.
  • the five types of proteins involved in de novo nucleosome assembly are conserved among evolutionarily distant organisms such as S. cerevisiae and humans .
  • CAC complex consisting of factors including but not lim- - ited to: CAF-1 pl5 . 0, CAF-1 p60, CAF-1 p48, 2.
  • the ASFl/HIR complex consisting of factors including but not limited to: ASFl, Hirl, Hir2, H ⁇ ?3,"Hpc2
  • Asfl is a small evolutionarily conserved acidic protein that also binds to newly synthesised H3 and H4 (Tyler et al . , Nature
  • HATs B-type histone acetyl transferases
  • HDACs histone deacetylases
  • chromatin assembly factor includes one or more components of Chromatin Assembly Factor-1 (CAFl, Verreault et al . , Cell 1996; 87: 95), a three-polypeptide protein that functions at the DNA replication fork via a direct interaction with the Proliferating Cell Nuclear Antigen (PCNA) , a DNA poly- merase processivity factor that forms a sliding clamp around DNA.
  • PCNA Proliferating Cell Nuclear Antigen
  • pl50 also named chromatin assembly factor 1 subunit A; CHAFlA
  • p60 also named chromatin assembly factor 1 subunit B; CHAFlB, MPP7 and MPHOSPH7
  • p48 subunits Preferred components are pl50(also named chromatin assembly factor 1 subunit A; CHAFlA)
  • p60 also named chromatin assembly factor 1 subunit B; CHAFlB, MPP7 and MPHOSPH7
  • p48 subunits are Preferred components.
  • CAF proteins can be identified in a large number of plants .
  • sequences from other plants which are homologous to the known or herein disclosed CAF proteins can be found readily for example by database searches or by screening genetic libraries using a CAF polypeptide sequence or CAF nucleic acid sequences as search sequence or probe.
  • CAF-1 proteins known in the art may include but shall not be limited to proteins from Arabidopsis thaliana (dbj : BAA77811.1; BAA77812.1; GenBank AB027230; AF016846; AF375435_1 AT5g64630; AAM47965; dbj BAA96914; GenBank AAL24356; GenBank AAB70242, SwissProt 022467; At5g58230) , rice--(dbj: BAC06268.1), tomato (Lycopersicon esculentum; GenBank ⁇ TFOl ' 6845; pir: T04324; SwissProt : .022466; ' GenBank AAB70241) ,• corn (Zea mays, GenBank AF440219, AAL33648.1, AF440219_1; pir T04324) ; Pinus pinaster (GenBank AL750826) ; Beta vulgaris (GenBank BI543305, BI54330
  • chromatin assembly factor other factors and components which facilitate, enable, or enhance function of the above mentioned CAF-1 proteins.
  • the term also includes the proliferating cell nuclear antigen (PCNA) .
  • PCNA proliferating cell nuclear antigen
  • CAF'-.I is recruited to sites of DNA synthesis via direct interaction with PCNA, the processivity factor for DNA polymerases (Moggs J et al. (2000) Mol Cell Biol 20:1290-1299).
  • the p60 subuni of CAP-I physically interacts with the Asflp/H3/H4 histone deposition complex.
  • PCNA proteins from plants are known and characterized (Lopez I et al.
  • CAF-1 pl50 protein is intended to include a naturally occurring protein encoded by an amino acid sequence comprising a sequence having a homology of at least 50%, preferably 70%, more preferably 90%, most preferably 100% to at least one of the following CAF-1 pl50 consensus sequence ⁇ motifs,-, (amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein "X” can stand for any amino acid. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes) : ⁇ " "
  • ALLXQQK b) ALXK(T/S)QPL(I/V)I c) NLTHEK - ' " > d) KEKLX(E/Q)E ' " - e) EXXKEXXEXEXR f) DEXEKXXKXRXKE g) LDYEVDSD(E/D)E EEEEXGESLS h) VPXGYLSEDEGV i) EEXXLPLSKLXDEIXXKL j) VGQRV . ' .,.
  • motifs a, b, c, g, and h are preferred. Especially preferably, at least two or three, preferably at least four or five, more ⁇ preferably at least six or seven, most preferably eight, nine or ten sequences having the respective homology to different motifs (a to j) occur in a CAF-1 pl50 protein. Further sequence motifs which are typical for CAF-1 pl50 can be deduced readily by the skilled worker from the sequence alignment of the known CAF-1 pl50 proteins, as shown in Fig. la-c.
  • CAF-1 pl50 proteins can alternatively be characterized as pro- teins comprising one or more of the following motifs :
  • TAF homology domain (1 site), Accession: IPB003894A, localization in A.thaliana CAF-1 pl50: aa 791-805 (Score: 1068.0)
  • HMG-I and HMG-Y DNA-binding domain (A+T-hook) : (1 site), Accession: IPB000637B, localization in A.thaliana CAF-1 pl50: 540-558 (Score: 1067.0) KDEDESLEEGCSKADDEDD KDEDESLEEGCSKADDEDD
  • KE2 family protein (1 site), Accession: IPB002777B, local- • ization in A.thaliana CAF-1 pl50: 294-330 (Score: 1067.0) kEKEeTESRkrIKKqqDesEKEQKRrEKegAELKKQL KEKEETESRKRIKKQQDESEKEQKRREKEQAELKKQL (correspond to CAFA-Human K/D/E-rich region Aa305-435) d) C-C chemokine receptor type 6 signature: (1 site) , Accession: PR01529C, localization in A.tha'T ⁇ ana CAF-1 pl50: 419-428 (Score: 1066.0)
  • EGF-like domain (1 site), Accession: IPB000561, localization in A.thaliana CAF-1 pl50: 487-495 (Score: 1050.0) ' ' KSCRPGFYG KSCRPGFYG
  • a CAF-1 pl50 protein comprise a "KE2 family protein" motif (motif c above) , which corresponda to CAFA-Human K/D/E-rich region (Aa305-435) .
  • CAF-1 pl50 proteins include proteins from the group consisting of:
  • a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
  • a functionally equivalent polypeptide molecule comprising an amino acid sequence which is at least 60%, preferably at least
  • a polypeptide molecule comprising a ' fragment of at least 20 consecutive amino acids, preferably 50 consecutive amino acids of at least one of the sequences described under a) or ,b) ex-.' 1 hibiting essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • "Property" or "properties" of a CAF ⁇ pl50 protein is to be understood in the broad sense and includes for example activity and/or function of said CAF-1 pl50 protein.
  • “Function” is pref- erably understood to mean the substrate-binding capacity or ligand-binding property of a CAF-1 pl50 polypeptide in an organism, a tissue, a cell or a cell compartment.
  • CAF-1 pl50 proteins are known to bind to several interaction partners including but not limited to CAF-1 p60, CAF-1 p48, and PCNA. The. binding ca- pacity with regard to this proteins is to be understood as an essential property of CAF-1 pl50 proteins.
  • Binding partners for CAF-1 pl50 proteins can be identified by methods well known to the person skilled in the art, for example by the yeast-2-hybrid system. Said interaction partners may include but shall not be limited to plant homologues of binding partners of CAF-1 p60, p48 or PCNA.
  • CAF-1 p60 (FAS2) protein is intended to include a naturally occurring protein encoded by an amino acid sequence comprising a sequence having a homology of at least 50%, preferably 70%, more preferably 90%, most preferably 100% to at least one of the following CAF-1 p60 consensus sequence motifs (amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein "X” can stand for any amino acid. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes) :
  • Further sequence motifs which are typical for CAF-1 p60 can be deduced readily by the skilled worker from the sequence alignment of the known CAF-1 p60 proteins, as shown in Fig. 2 and 3.
  • CAF-1 p60 proteins can alternatively be characterized as proteins comprising one or more of the following motifs:
  • G-protein beta WD-40 repeats (1 site), Accession: IPB001680; localization in A.thaliana CAF-1 p60 196-207 (Score ' : 1 - 1124.0) SGSDDAQICLWD SGSDDAQICLWD
  • G-protein beta WD-40 repeats (1 site) Accession: IPB001680 localization in A.thaliana CAF-1 p60 291-302 (Score: 1098.0) TGSTDKTVKLFD TGSTDKTVKLFD
  • Gag gene protein p24 (core nucleocapsid protein) : (1 site) ; Accession: IPB000721A; localization in A.thaliana CAF-1 p60 19-29 (Score: 1072.0) EEYKIWKKNTP EEYKIWKKNTP
  • G-protein beta WD-40 repeats (1 site); Accession: IPB001680; localization in A.thaliana CAF-1 p60: 245-256 (Score: 1065.0)
  • G-protein beta WD-40 repeats (1 site); Accession: IPB001680; localization in A.thaliana CAF-1 p60: 335-346 (Score: 1029.0) SCCLGRRLMVWD SCCLGRRLMVWD
  • CAF-1 p60 proteins include proteins from the group consisting of:
  • a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 50, 52, 54, " ⁇ 56, or 58;
  • a functionally equivalent polypeptide molecule comprising an amino acid sequence which is at least 60%, preferably at east 80%, by preference at least 90%, especially preferably at least 95%, very especially preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 50, 52, 54,.' ' -56, or 58 and exhibits essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 50, 52, 54, 56, or 58; and
  • polypeptide molecule comprising a fragment of at least 20 consecutive amino acids, preferably 50 consecutive amino acids of at least one of the sequences described under a) or b) exhibiting essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 50, 52, 54, 56, or 58.
  • “Property” or “properties” of a CAF-1 p60 protein is to be understood in the broad sense and includes for example activity and/or function of said CAF-1 p60 protein.
  • “Function” is preferably understood to mean the substrate-binding capacity or ligand-binding property of a CAF-1 p60 polypeptide in an' organism, a tissue, a cell or a cell compartment.
  • CAF-1 p60 proteins are known to bind to several interaction partner including but not limited to CAF-1 pl50, CAF-1 p48, and PCNA. The binding capacity with regard to this proteins is to be understood as an essential property of CAF-1 p60 proteins.
  • Binding partners for CAF-1 p60 proteins can be identified by methods well known to the person skilled in the art, for example by the yeast-2-hybrid system. Said interaction partners may include but shall not be limited to plant homologues of binding partners of CAF-1 pl50, p48, or PCNA.
  • CAF-1 p48 protein is intended to include a naturally occurring protein encoded by an amino acid sequence comprising a sequence having a homology of at least 50%, preferably 70%, more preferably 90%, most preferably 100% to at least one of the following CAF-1 p48 consensus sequence motifs (amino acids may be referred to hefSln ' by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein "X” can stand for any amino acid. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes) : ⁇
  • motifs a, b, d, and h are preferred. Especially preferably, at least two or three, preferably at least four or five, more pref- erably at least six or seven, most preferably eight sequences having the respective homology to different motifs (a to h) occur in a CAF-1 p48 protein. Further sequence motifs which are typical for CAF-1 p48 can be deduced readily by the skilled worker from the sequence alignment of the known CAF-1 p48 pro- teins, as shown in Fig. 4.
  • CAF-1 p48 proteins include proteins from the group consisting of:
  • a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 38, 40, 42, 44, 46, or 48;
  • a functionally equivalent polypeptide molecule comprising an amino acid sequence which is at least 60%, preferably at least
  • polypeptide molecule comprising a fragment of at least 20 consecutive amino acids, preferably 50 consecutive amino acids of at least one of the sequences ⁇ c-escribed under a) or b) exhibiting essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 38, 40, 42, 44, 46, or 48.
  • “Property” or “properties” of a CAF-1 p48 protein is to be un- ⁇ derstood in the broad sense and includes for example activity and/or function of said CAF-1 p48 protein.
  • “Function” is pref- erably understood to mean the substrate-binding capacity or ligand-binding property of a CAF-1 p48 polypeptide in an organism, a tissue, a cell or a cell compartment.
  • CAF-1 p48 proteins are known to bind to several interaction .partner including but not limited to CAF-1 pl50, CAF-1 p60, and PCNA. The binding ca- pacity with regard to this proteins is to be understood as an essential property of CAF-1 p60 proteins.
  • Binding partners for CAF-1 p48 proteins can be identified by methods well known to the person skilled in the art, for example by the yeast-2-hybrid system. Said interaction partners may include but shall not be limited to plant homologues of binding partners of CAF-1 pl50, p60, or PCNA.
  • PCNA PCNA protein
  • PCNA PCNA protein
  • amino acid sequence comprising a sequence having a homology of at least 50%, preferably 70%, more preferably 90%, most preferably 100% to at least one of the following PCNA consensus sequence 'motifs (amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recom- mended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein "X” can stand for any amino acid. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes) :
  • motifs a, b, c, d, ancF f 1 Especially preferably, at least two or three, preferably at least four or five, more preferably at least six or seven, most preferably eight, nine or ten sequences having the respective homology to different motifs (a to j) occur in a PCNA protein. Further sequence motifs which are typical for PCNA can be deduced readily by the skilled worker from the sequence alignment of the known PCNA proteins, as shown in Fig. 5.
  • PCNA proteins can alternatively be characterized as proteins, comprising one or more of the following motifs: a) Proliferating cell nuclear antigen: (1 site) ; Accession: IPB000730A; localization -in A.thaliana PCNA: 24-78. ' ( : Score: 1525.0)
  • IPB000730C localization in A.thaliana PCNA: 197-250 (Score: 1372.0) IEMKEPVSLSFALRYMNSFTKATPLSDTVTISLSSELPVWEYKVAEMGYIRYY IEMKEPVSLSFALRYMNSFTKATPLSDTVTISLSSELPVVVEYKVAEMGYIRYY
  • PCNA proteins include proteins from the group consisting of:
  • a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, or 36;
  • a functionally equivalent polypeptide molecule comprising an - amino acid sequence which is at least 60%, preferably at least 80%, by preference at least 90%, especially preferably at least 95%, very especially preferably at least 98% identical ., to the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, or 36 and exhibits essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 22, 24, 26., 28 , 30 , 32 , 34 , or 36 ; and
  • polypeptide molecule comprising a fragment of at least 20 consecutive amino acids, preferably 50 consecutive amino acids of at least one of the sequences described under a) or b) exhibiting essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, or 36.
  • PCNA proteins are known to bind to several interaction partner including but not limited to DNA, CAF-1 ' pl50, CAF-1 p60, and CAF-1 p48. The binding capacity with regard to this proteins is to be understood as an essential property of PCNA proteins . Binding partners for PCNA proteins can be identified by methods well known to the person skilled in the art, for example by the yeast-2-hybrid system. Said interaction partners may include but shall not be limited to plant homologues of binding partners of CAF-1 pl50, p60, and p48.
  • “Functional equivalent polypeptide” is understood to mean, in particular, natural or artificial mutations of a CAF polypeptides (e.g. encoded by a polypeptide comprising a sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58) and homologous polypeptides from other eukaryotic species, preferably vascular plants, which continue to have essentially the same properties. Homologous polypeptides from the below-described preferred vascular plants are preferred. Mutations encompass substitutions, additions, deletions, inversions or insertions of one or more amino acid residues.
  • the present invention also encompasses those polypeptides which are obtained by modification of. a polypeptide comprising a sequence as -shown ' _ in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, ' 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58.
  • Preferred functional equivalents include conservatively- modified variants of the CAF proteins (e.g. encoded by a polypeptide comprising a sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58).
  • Constantly modified variants applies to both amino acid and nucleic acid sequences.
  • conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al . , Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al . , Mol. Cell. Probes 8: 91-98 (1994)). Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are "silent variations", which are one species of conservatively modified variations .
  • Every nucleic acid sequence recited herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except , ⁇ « AUG, which, along with GUG in some organisms, is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic a- cid, peptide, polypeptide, or protein sequence which alter, adc or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified vari- ant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and al- leles of the invention.
  • another embodiment of the invention comprises an iso- lated polypeptide sequence coding for a CAF protein, wherein said polypeptide sequence comprises an amino acid sequence described by SEQ ID NO: 12, 14, 16, 18, 20, 46, 48, 56, or '58.
  • a further embodiment of the invention comprises an isolated nu- ' cleic acid sequence coding for a CAF protein of the invention, wherein preferably said nucleic acid sequence comprises an sequence described by SEQ ID NO: 11, 13, 15, 17, 19, 45, 47, 55, or 57.
  • sequences from other plants which are homologous to the CAF sequences disclosed within the scope of the present invention can be found readily for example by database searches or by screening genetic libraries using the CAF sequences as search sequence or probe.
  • Additional CAF proteins can be identified for example from a variety of organisms for which DNA sequences are known, such as, for example, from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Oryza sativ , or . Helianthus annuus from databases or homology comparisons .
  • the probes derived from the nucleic acid sequences as shown in SEQ ID NO: 1, 3, 57* 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57 have a length of at least 20 bp, preferably 50 bp, particularly preferably 100 bp, very especially preferably 200 bp, and most preferably 400 bp.
  • a DNA strand which is complementary to the sequences described under SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, '41, 43, 45, 47, 49, 51, 53 55, or 57 may also be employed for screening the libraries.
  • Functional equivalents accordingly, encompass DNA sequences which hybridize under standard conditions with the CAF nucleic acid sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, with the sequence complementary thereto or parts of the above entioned and which, as complete sequences, encode proteins which have the same properties as the proteins described under SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,. or 58.
  • the method of the invention may be applied to all eukaryotic cells and organism, preferably to cells derived from or able to form a pluricellular organism and/or which are recalcitrant to transformation and/or homologous recombination.
  • the method of the invention can be carried out by decreasing the expression, amount of protein, amount of RNA or mRNA, function, activity of a chromatin assembly factor, preferably a CAF-1 protein, more preferably a CAF-1 pl50 protein.
  • Amount of protein is understood as meaning the amount of protein (e.g., an CAF polypeptide) in an organism, a tissue,,, a cell, or a cell compartment.
  • a decrease in the amount of protein (and indirectly the encoding mRNA) may be monitored by various techniques well known to the person skilled in the art including but not limited to the micro-biuret method (Goa J (1953) Scand J Clin Lab Invest 5 :218-222) , the Folln-Ciocalteu-method (Lowry OH et al.
  • “Decrease” with respect to the amount of mRNA or protein is to be understood in the broad sense and is intended to include .the permanent or temporarily, partially, essentially completely or completely hindering or blocking of the expression of the target gene or the corresponding RNA or mRNA by various cellular mecha- nisms or of the protein product resulting thereof.
  • a decrease in the amount of mRNA may be monitored by various techniques well known to the person skilled in the art including but not limited to Northern-hybridization, nuclease protection assay or quantitative reverse transcription PCR (quantitative RT-PCR) .
  • RNA or mRNA or protein means the quantitative decrease of the amount of RNA or mRNA or protein, respectively, in an organism, a tissue, a cell or a cell compartment in comparison with the wild type of the same genus and species, to which this method had not been applied, under otherwise identical conditions (such as, for example, culture conditions, plant age and the like) .
  • the decrease amounts to at least 10%, preferably at least 10% or at least 20%, especially preferably at least 40% or 60%, very especially preferably at least 70% or 80%, most preferably at least 90% or 95%.
  • a complete inactivation of expression and/or gene function is preferred ("knock-out").
  • Decrease with respect to the function and/or activity of a protein (e.g., expressed from a target gene or coding for a CAF protein) may include an decrease of the encoding mRNA but may also include a change in the protein sequence of the gene product causing a decreased or abolished function and/or activity.
  • a decrease also encompasses a quantitative decrease of a chromatin assembly factor down to the essential complete absence of said chromatin assembly factor
  • chromatin assembly factor (i.e. lacking detectability of the function or immunologies! de- tectability of said chromatin assembly factor) .
  • expression of a particular chromatin assembly factor, or its activity or function, in a cell or an organism is preferably re- prised by more than 50%, especially preferably by more than 80%, very especially preferably by more than 90%.
  • a variety of strategies for reducing the expression of chromatin assembly factor, its activity or function are encompassed in ac- cordance with the invention.
  • the skilled worker is aware of a series of different methods being available for influencing the expression of a chromatin assembly factor, its activity or function in the desired manner.
  • a decrease of the chromatin assembly factor activity or function is preferably achieved by reduced expression of an endogenous chromatin assembly factor.
  • a decrease of the amount of chromatin assembly factor, its expression, activity or function can be effected - for example - by utilization of at least one of the following methods:
  • RNA a double-stranded RNA of a chromatin assembly factor encoding nucleic acid sequence (CAF protein dsRNA) or an expression cassette (s) ensuring the expression thereof
  • RNA of a chromatin assembly factor encoding nucleic acid sequence or an expression cassette ensuring expression thereof.
  • the antisense nucleic acid sequence is directed against a chromatin assembly factor gene (i.e. genomic DNA sequences) or a chromatin assembly factor gene transcript (i.e. RNA sequences) .
  • ⁇ -anomeric nucleic acid sequences are also encompassed
  • the sense nucleic acid " sequence may be DNA or RNA, but is preferably RNA.
  • each and every one of these methods may bring about a decrease of the expression of a CAF protein, activity and/or function of a CAF protein for the purposes of the -invention.
  • a combined use is also feasible.
  • Further methods are known to the skilled worker and can encompass the hindering or preven- tion of CAF protein processing, of the CAF protein or CAF mRNA transport, inhibition of ribosome attachment, inhibition of RNA splicing, induction of an CAF protein RNA-degrading enzyme and/or inhibition of translational elongation or termination.
  • dsRNAi double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • dsRNAi methods are based on the phenomenon that the simultaneous introduction of complementary strand and counterstrand of a gen*e transcript causes the expression of the gene in question to be suppressed in a highly efficient manner. The phenotype caused greatly resembles a corresponding knock-out mutant (Waterhouse PM et al. (1998) Proc Natl Acad Sci USA 95:13959-64).
  • dsRNAi has proved to be particularly effective and advantageous for reducing expression of a CAF protein.
  • dsRNAi approaches are markedly superior to traditional antisense approaches .
  • the invention therefore furthermore relates to double-stranded RNA molecules (dsRNA molecules) which, upon introduction into a plant (or a cell, tissue, organ or seed derived therefrom) , bring about a decrease of expression of a CAF protein.
  • dsRNA molecules double-stranded RNA molecules
  • Double-stranded RNA molecule is to be understood to comprise at least one RNA molecule, which is at least theoretically able to form a double-stranded RNA secondary structure by intermolecular or intramolecular base pairing.
  • Such secondary structure may be predicted by the base-paring rules of Watson and Crick or by computer algorithms (like e.g., FOLDRNA; Zuker and Stiegler (1981) Nucleic Acids Res 9 (1) : 133-48) .
  • the double-stranded RNA molecule for reducing the expression of an CAF protein comprises,
  • RNA sequence which is essentially iden- tical to at least part of a nucleic acid sequence coding for a CAF protein
  • RNA sequence which is essentially complementary to at least part of said first RNA sequence un- der i) .
  • the double-stranded RNA molecule comprises,
  • RNA sequence which is essentially iden- tical to at least part of a nucleic acid sequence compris- ing a sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 2 ⁇ 29, 31, 33, 35, 37, 39, 41, 43., 45, 47, 49, 51, 53, 55, or 57, and
  • RNA sequence which is essentially com- -plementary to at least part .of said first RNA sequence under i) . • -
  • dsRNA sequence can also show insertions, deletions or individual point mutations compared with the target sequence coding for the CAF protein while still bringing about an effective decrease of the expression.
  • the homology in accordance with the above definition preferably amounts to at least 75%, preferably at least 80%, very ' ⁇ espe- cially preferably at least 90%, most preferably 100%, between the sense strand of an inhibitory dsRNA and a part-segment of a nucleic acid sequence encoding a CAF protein (or between the antisense strand and the complementary strand of a nucleic acid sequence encoding a CAF protein) .
  • the length of the part-segment amounts to at least 10 bases, preferably at least 25 bases, especially preferably at least 50 bases, very especially preferably at least 100 bases, most preferably at least 200 bases or at least 300 bases.
  • an "essentially identical" dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing with part of a CAF gene transcript (for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at -50°C or 70°C for 12 to 16 h) .
  • dsRNA molecules in the methods for introducing mutations into the chromosomal DNA of a plant cell- or a plant organism.
  • the dsRNA can be composed of one or more strands of polymerized ribonucleotides . Modifications both of the sugar-phosphate backbone and of the nucleosides may be present. For example, the phosphodiester bonds of the natural RNA can be modified in such a way that they comprise at least one nitrogen or sulfur hetero ⁇ atom. Bases can be modified i such a way that the activity of, for example, adenosine deaminase is restricted. These and other modifications are described hereinbelow in the methods of stabi- ' lizing antisense RNA. The dsRNA can be generated enzymatically or fully or partially synthesized chemically.
  • the double-stranded structure can be formed starting from an individual self-complementary strand' ⁇ r starting from two complementary strands.
  • sense and antisense sequence may be linked by a linking sequence ("linker") and can form for example a hairpin structure.
  • the linking sequence can preferably be an intron which is spliced out after the dsRNA has been synthesized.
  • the nucleic acid se- '"4 quence encoding a dsRNA can comprise further elements such as, for example, transcription termination signals or polyadenyla- tion signals.
  • RNA sequences are to be combined to form a dsRNA in a cell or plant, this can be effected in various ways:
  • the formation of the RNA duplex can be initiated either outside or within the cell.
  • the dsRNA can also en- compass a hairpin structure by linking sense and antisense strand by means of a linker (for example an intron) .
  • a linker for example an intron
  • the self- complementary dsRNA structures are preferred since they only require the expression of one construct and always comprise the complementary strands in an equimolar ratio .
  • the expression cassettes encoding the antisense or sense strand of a dsRNA or the self-complementary strand of the dsRNA are preferably inserted into a vector and, using the methods described hereinbelow, stably inserted Into the genome of a plant in order to ensure permanent expression of the dsRNA, using selection markers for example. ,.,
  • the dsRNA can be introduced using a quantity which allows at least one copy per cell. Greater quantities (for example at least 5, 10, 100, 500 or 1000 copies per cell) may bring about a more effective decrease.
  • Fig.l to.5 allows the conclusion that this protein is conserved to a high degree within plants, so that the expression of a dsRNA derived from one of the disclosed nucleic acid sequences encoding a CAF protein as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 appears to have an advantageous effect in other plant species as well .
  • the dsRNA can be synthesized either in vivo or in vitro.
  • a DNA sequence encoding a dsRNA can be brought into an expression cassette under the control of at least one genetic con- trol element (such as, for example, promoter, enhancer, silencer, splice donor or splice acceptor or polyadenylatipn signal) .
  • at least one genetic con- trol element such as, for example, promoter, enhancer, silencer, splice donor or splice acceptor or polyadenylatipn signal.
  • Suitable advantageous constructions are described herein- below. Polyadenylation is not required, nor do elements for initiating translation have to be present .
  • a dsRNA can be synthesized chemically or enzymatically.
  • Cellular RNA polymerases or bacteriophage RNA polymerases (such as, for example, T3, T7 or SP6 RNA polymerase) can be used for this purpose.
  • Suitable methods for expression of RNA in vitro are de- scribed (WO 97/32016; US 5,593,874; US 5,698,425, US 5,712,135, US 5,789,214, US 5,804,693).
  • a dsRNA which has been synthesized in vitro chemically or enzymatically can be isolated completely or to some degree from the reaction mixture, for example by extraction, precipitation, electrophoresis, chromatography or co - binations of these methods, before being introduced into a cell, tissue or organism.
  • the dsRNA can be introduced directly , technicallyinto .> « . the cell or else be applied extracellularly (for example into the interstitial space) .
  • the antisense nucleic acid molecule hybridizes, or binds, with the cellular mRNA and/or genomic DNA encoding the CAF target protein to be suppressed. This suppresses the transcription
  • Hybridization can originate conventionally by the formation of a stable duplex or - in the case of genomic DNA - by the antisense nucleic acid molecule binding to the duplex of the genomic DNA by specific interaction in the major groove of the DNA helix.
  • An antisense nucleic acid sequence suitable for reducing an CAF protein can be deduced using the nucleic acid sequence encoding this protein, for example the nucleic acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
  • the antisense nucleic acid sequence can be complementary to all of the- transcribed mRNA of said protein, be limited to the coding region', or else only be composed of a nucleotide, which is partially
  • the oligonucleotide can be complementary to the region encompassing the translation start for said protein.
  • Antisense nucleic acid sequences can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length, but may also
  • Antisense nucleic acid sequences can be expressed recombinantly or synthesized chemically or enzymatically
  • Modified nucleotides can impart an increased biochemical stability to the antisense nucleic acid sequence and lead to an in- ,- « creased physical stability of the duplex formed of antisense nucleic acid sequence and sense target sequence.
  • the following can be. used: for example phosphorothioate derivatives or acridine- substituted nucleotides.
  • the expression of a CAF protein can be inhibited by nucleotide sequences which are comple- mentary to the regulatory region of a CAF gene (for example a CAF promoter and/or enhancer) and which form triple-helical structures with that DNA double helix so that the transcription * of the CAF gene is reduced.
  • nucleotide sequences which are comple- mentary to the regulatory region of a CAF gene (for example a CAF promoter and/or enhancer) and which form triple-helical structures with that DNA double helix so that the transcription * of the CAF gene is reduced.
  • the antisense nucleic acid molecule can be an -anomeric nucleic acid.
  • Such ⁇ -anomeric nucleic ' 'acid molecules form specific double-stranded hybrids with complementary RNA in which - as opposed to the conventional ⁇ -nucleic acids - the two strands run parallel to one another (Gautier C et al. (1987) Nucleic Acids Res 15:6625-6641).
  • the antisense nucleic acid molecule can furthermore also comprise 2 ' -0- methylribonucleotides (Inoue et al. (1987) Nucleic Acids Res 15:6131-6148) or chimeric RNA/DNA analogs (Inoue et al. (1987) FEBS Lett 215:327-330).
  • RNA molecules or ribozymes can be adapted to suit any target RNA and cleave the phosphodiester backbone at specific positions, functionally deactivating the target RNA (Tanner NK (1999) FEMS Microbiol Rev 23 (3) :257-275) .
  • the ribozyme itself is not modified thereby, but is capable of cleaving further target RNA molecules analogously, thereby assuming the qualities of an enzyme.
  • ribozyme sequences into antisense RNAs confers this enzymelike RNA-cleaving quality to precisely these antisense RNAs, thus increasing their efficacy in inactivating the- target RNA.
  • the generation and the use of such ribozyme antisense RNA mole-- cules is described, for example, in Haseloff et al . (1988) Na- ture 334: 585-591.
  • ribozymes for example "hammerhead” ribozymes; Haselhoff and Gerlach (1988) Nature 334:585-591
  • the ribozyme tech- nique can increase the efficacy of " an antisense strategy.
  • Methods of expressing ribozymes for recTucihg specific proteins are described in (EP 0 291 533, EP 0 321 201, EP 0 360 257). The expression of ribozyme in plant cells has also been described (Steinecke P et al .
  • Suitable target sequences and ribozymes can be determined as described for exa ⁇ rt- ple by "Steinecke P, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds, Academic Press, Inc. (1995), pp. 449-460" by calculating the secondary structure of ribozyme RNA and target RNA as well as by their interaction (Bayley CC et al. (1992) Plant Mol Biol. 18 (2) :353-361; Lloyd AM and Davis RW et al .
  • the expression of an CAF nucleic acid sequence in sense orienta- tion can lead to cosuppression of the corresponding homologous endogenous gene.
  • the expression of sense RNA with homology with an endogenous gene can reduce or switch off the expression of the former, similarly to what has been described for antisense approaches (Jorgensen et al. (1996) Plant Mol Biol 31(5) :957- 973; Goring et al . (1991) Proc Natl Acad Sci USA 88:1770-1774; Smith et al. (1990) Mol Gen Genet 224:447-481; Napoli et al . (1990) Plant Cell 2:279-289; Van der Krol et al. (1990) Plant Cell 2:291-99).
  • homologous gene to be reduced can be repressed either fully or only in part by the con- struct introduced.
  • the possibility of translation is not required.
  • the application of this technique to plants is described, for example, by Napoli et al. (1990) The Plant Cell 2: 279-289 and in US 5,034,323.
  • CAF gene expression may also be reduced using specific DNA- binding factors, for example factors of the zinc finger tran- scription factor type. These factors attach to the genomic se- quence of the endogenous target gene, preferably in the regulatory regions, and bring about repression of the endogenous gene.
  • the use of such a method makes possible the decrease of the expression of an endogenous CAF gene without it being necessary to recombinantly manipulate its sequence.
  • suitable methods for the preparation of suitable factors have been described (Dreier B et al. (2001) J Biol Chem 276 (31) :29466-78; Dreier B et al. (2000 J Mol Biol 303(4) :489-502; Beerli RR et al.
  • CAF gene This segment is preferably located in the promoter region. For gene suppression, however, it may also be in the region of the coding exons or introns .
  • the segments in question can be obtained by the skilled worker from GeneBank by database search or, starting from an CAF cDNA whose gene is not present in GeneBank, by screening a genomic library for corresponding genomic clones . The skilled worker is familiar with the methods required therefore.
  • the protein-binding factors can be, for example, aptamers (Famulok M and Mayer G (1999) Curr Top Microbiol Immunol 243:123-36) or antibodies or antibody fragments or single-chain antibodies . Methods for ob- taining these factors have been described and are known to the skilled worker.
  • a cytoplasmic scFv antibody was employed to modulate the activity of the phytochrome A protein in genetically modified tobacco plants (Owen M et al. (1992). Bio-' technology (N Y) 10 (7) :790-794; Franken E et al . (1997) Curr Opin Biotechnol 8 (4) :411-416; Whitelam (1996) Trend Plant Sci 1:286-272).
  • Gene expression may also be suppressed by tailor-made low- molecular-weight synthetic compounds, for example of the polyam- ide type (Dervan PB and B ⁇ rli RW (1999) Current Opinion in Chemical Biology 3:688-693; Gottesfeld JM et al. (2000) Gene Expr 9 (1-2) :77-91) .
  • Inhibition of CAF expression can also be brought about effi- ciently by inducing the specific CAF RNA degradation by the plant with the aid of a viral expression system (amplicon) (An- gell, SM et al . (1999) Plant J. 20 (3) : 357-362) .
  • amplicon a viral expression system
  • VGS viral induced gene silencing
  • nucleic acid construct comprising at least part of an endogenous CAF gene which is modified by a deletion, addition or substitution of at least one nucleotide in such a way that its functionality is reduced ,• « or fully destroyed.
  • the modification may also relate to the regulatory elements (for example the promoter) of the gene, so that the coding sequence remains unmodified, but expression (transcription and/or translation) does not take place or is re- prised.
  • the modified region is flanked at its 5' and 3' end by further nucleic acid sequences which must be sufficient in length for making possible recombination. They are, as a rule, in the range of several hundred bases to several kilobases in length (Thomas KR 1 and Capecchi MR (1987) Cell -51:503 ,- Strepp et al. (1998) Proc Natl Acad Sci USA 95 (8) :4368-4373) .
  • the host organism - for example a plant - is transformed with the recombination construct using the methods described, hereinbelow, and clones which have successfully undergone recombination are selected, for example using. a resistance to antibiotics or herbicides . ⁇ • ,' ⁇
  • Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations into the host genome predominate.
  • One possibility of eliminating the randomly integrated sequences and thus increasing the number of cell clones with a correct homologous recombination is the use of a sequence-specific recombination system as described in US 6,110,736, by which unspecifically integrated sequences can be deleted again, which simplifies the selection of events which have integrated successfully via homologous recombination.
  • a large number of sequence-specific recombination systems can be used, examples being the Cre/lox system of bacteriophage Pi, the FLP/FRT system of yeast, the Gin recombinase of phage Mu,- the Pin recombinase from E.coli, and the R/RS system of the pSRl ' plasmid.
  • the bacteriophage PI Cre/lox and the yeast FLP/FRT sys- tem are preferred.
  • the FLP/FRT and cre/lox recombinase system has already been applied to plant systems (Odell et al . (1990) Mol Gen Genet 223: 369-378).
  • RNA/DNA oligonucleotides into the plant
  • knock-out mutants with the aid of, for example, T- DNA mutagenesis
  • mutations may in induced by transposon mediated mutagenesis (Sundaresan V et -al. (1995) Genes Dev. 9 (14) : 1797-810; Parinov S et al. (1999) Plantf Cell 11(12) :2263-70) .An additional method for inducing mutations in endogenous gene may utilize "Targeting Induced Local Lesions IN Genomes” (TILLING) (McCallum CM (2000) Plant Physiology 123:439-442).
  • Mutations resulting in a dominant negative phenotype may affect binding of pl50CAFl to p60CAFl or to PCNA. When expressed in cells, dominant negative pl50CAFl disrupts the endogenous CAFl complex. Mutants affecting pl50 CAF functionality and resulting in a dominant negative phenotype may be localized in the PCNA binding region or the Cac3p (CAF-1 p48) binding region.
  • F233L and F233A/F234G mutants which demonstrate a deficiency in PCNA binding
  • F257F and/or L276P mutants demonstrating a impaired Cac3p (CAF-1 p48) binding.
  • PTGS post- transcriptional gene silencing
  • the person skilled in the art may easily identify equivalent ways to achieve the same effect than achieved by decreasing ac- tivity and/or expression of a CAF protein, e.g., by decreasing activity and/or expression of interaction partners of the CAF protein or other proteins which participate in the same signal transduction pathway than the CAF protein.
  • Such proteins may be identified using methods known to the person skilled in the art to elucidate signal transduction pathways like., e.g. interaction partner network analysis utilizing yeast-two-hybrid or yeast-n-hybrid systems .
  • anti-CAF anti-CAF compounds
  • the term "anti-CAF” compound explicitly includes the nucleic acid sequences, peptides, proteins or other factors employed in the above-described methods.
  • introduction comprises all of the methods which are capable of directly or indirectly introducing an "anti-CAF” compound into a plant or a cell, com- partment, tissue, organ or seed thereof, or of generating such a compound there.
  • Direct and indirect methods are encompassed.
  • the introduction can lead to a transient presence of an "anti-CAF” compound (for example a dsRNA) or else to its stable presence.
  • the "anti-CAF” compound can exert its function directly (for example by insertion into an endogenous CAF gene) .
  • its function can also be exerted indirectly following transcription into an RNA (for example in the case of antisense approaches) or following transcription and translation into a protein (for example in the case of binding factors) .
  • the invention encpmpasse-s both directly and indirectly acting "anti-CAF" compounds.
  • Any standard method known in the art may be utilized to intro- prise the DNA modifying reagents and/or recombination-inducing reagents into the recipient cells.
  • Such methods include, but are not limited to, microinjection, el ⁇ c'trbporation, passive adsorption, calcium phosphate-DNA co-precipitation, DEAE-dextran- mediated transfection, polybrene-mediated transfection, liposome fusion, lipofectin, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.
  • Particular cell types may be transfected with greater efficiency by one or more known methods. Processes of determining optimal conditions for transfection for a particular cell type are well known in the art, are practiced regularly and may be determined without undue experimentation.
  • introducing encompasses for example methods such as transfection, transduction or transformation. . '" --
  • Anti-CAF compounds therefore also encompasses recombinant expression constructs which bring about expression (i.e. transcription and, if appropriate, translation), for example of an CAF dsRNA or an CAF “antisense” RNA, preferably in a plant or a part, tissue, organ or seed thereof.
  • the expression construct of the invention comprise at least one nucleic acid molecule selected from the group consisting of:
  • nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57;
  • nucleic acid molecule comprising a fragment of at least 20 consecutive nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57;
  • nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least 60% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, ' 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58;
  • nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, " 56, or 58;, wherein the fragment comprises- at least 10 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,
  • chromatin assembly factors like, e.g., CAF-1 proteins, ⁇ were identified in numerous eukaryotic species and cells derived therefrom, the methods according to the invention can be applied to all eukaryotes, preferably to pluricellular eukaryotes or eukaryotic cells derived from or able to form said pluricellular organism.
  • the term "pluricellular organism” is intended to comprise an organism formed by two or more cells. Included are cells of human, non-human animal, fugal, and plant origin. Espe- cially preferred are plant species and cells derived therefrom..
  • non-human animal refers to any animal which is not a human and includes vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, capri- nes, equines, canines, felines, aves, etc. Preferred non-human animals are selected from the order Rodentia. "Non-human animal "additionally refers to amphibians (e. g. Xenopus), reptiles, insects (e. g. Drosophila) and other non-mammalian animal species.
  • amphibians e. g. Xenopus
  • reptiles e. g. Drosophila
  • insects e. g. Drosophila
  • Such cells are exemplified by embryonic cells (e. g. , oocytes, sperm cells, embryonic stem cells, callus cells, etc.), adult cells (e.'g., brain cells, fruit cells etc.), undifferentiated cells (e. g., ' fetal cells, tumor cells, etc.), differentiated cells (e. g., skin cells, lung cells, neural cells, muscle cells, blood cells, T cells, B cells, etc.), dividing cells, senescing cells, cultured cells, and the like.
  • the target cells may be primary cells or cultured cells.
  • a "primary cell” is a cell which is directly obtained from a tissue or organ of an animal in the absence of culture.
  • a primary cell is capable of undergoing ten or fewer passages in in vitro culture before senescence and/or cessation of proliferation.
  • a "cultured cell” is a cell which has been maintained and/or propagated in vitro. Cultured cells include "cell lines", i.e., cells which are capable of a greater number of passages in vitro before cessation of proliferation and/or ; « . senescence as compared to primary cells from the same source.
  • the cells are human and are exem- plified, but not limited to, U937 cells (macrophage) , ATCC# crl 1593.2; HUV-EC-C cells (vascular efidothelium) , ATCC# CRL-1730; ' Raji cells (lymphoblastoid) , ATCC# ⁇ 01-86; and HeLa cells (cervical carcinoma), ATCC# ccl-2.
  • the cells are non-human and are exemplified, but not limited to, LM cells (mouse fibroblast) , ATCC# ccl-1. 2; and BHK-21 cells (golden hamster kidney), ATCC# ccl-10. " '' .
  • the cells are capable of generating an animal .
  • Such cells are exemplified by, but not li- mited to, fertilized egg cells which may be implanted into the uterus of a pseudo-pregnant female and allowed to develop into an animal. These cells have successfully been used to produce transgenic mice, sheep, pigs, rabbits and cattle [Hammer et al. (1986) J Animal Sci 63 : 269; • Hammer et al . (1985) Nature 315:680- 683].
  • Other cells include pre-implantation embryo cells. For example, blastomere cells (Jaenisch, (1976) Proc Natl Acad Sci USA 73:1260-1264; Jahner et al .
  • ES cells are pluripotent cells which maybe directly derived from, for example, the inner cell mass of blastocysts (Doetchman et al. (1988) Dev Biol 127:224-227), from disaggregated morulae (Eistetter (1989) Dev Gro Differ 31:275- 282] or from primordial germ cells (Matsui et al. (1992) Cell 70:841-847).
  • Transgenic mice may be generated from ES cells which have been treated in accordance with the invention's methods by injection of several ES cells into the blastocoel cavity of intact blastocysts (Bradley et al . (1984) Nature 309: 225256) .
  • a clump of ES cells may be sandwiched between two eight-cell embryos [Bradley et al., (1987) in "Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,” Ed. Robertson E. J. (IRL, ' Oxford, U. K. ) , pp. 113- 151; Nagy et al . , (1990) Development 110: 815-821]. Both methods result in germ line transmission at high frequency. )A ,.,
  • the method of the invention is applied to plant cells.
  • plant is generally understood as meaning any single- or multi-celled organism or a eeli tissue, part or propagation material (such as seeds or fruit) of same which is capable of photosynthesis . Included for the purpose of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred. Also included are mature plants, seeds, ⁇ shoots and seedlings, and parts, propagation material (for example tubers, seeds or fruits) and cultures derived from them, for example cell cultures or callus cultures.
  • the term includes the mature plants, seed, shoots and seedlings and their derived parts, propagation material (such as seeds or microspores) , plant organs, tissue, protoplasts, callus and other cultures, for example cell cultures, and any other type of plant ' - ' -cell grouping to give functional or structural units.
  • Mature plants refers to plants at any desired developmental stage beyond that of the seedling. Seedling refers to a young immature plant at an early developmental stage.
  • plant organisms for the purposes of the invention are further organisms capable of being photosynthetically active such as, for example, algae, cyanobacteria and mosses.
  • Preferred algae are green algae such as, for example, algae from the genus Haematococcus , Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis is particularly preferred.
  • vascular plants are especially preferred.
  • the term "vascular plant” is intended to include all plants comprising' a vascular system in contrast to non-vascular plants like, e.g. Bryophytae.
  • Vascular plants may comprise seedless and seed- carrying vascular plants .
  • “Seedless vascular plants” are especially plants of the phyla Psilotophyta, Lycophyta, Sphenophyta or Pterophyta
  • Seed-carrying vascular plants are especially plants of the phyla Cycadophyta, Ginkophyta, Coniferophyta, Gnetophyta, Anthophyta.
  • Vascular plant encompasses all annual and perennial monocotyledonous and dicotyledonous plants and includes by way of exam-.. pie but not by limitation those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Tri- folium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsi- cum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis,- Majorana, Cichorium, ' "Helianthus, Lactuca, Bro- mus, Asparagus, ' Antirrhinum, Hemerocallis, Nemesis, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browall
  • Preferred plants are those from the following plant families : Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Llliaceae, Linaceae, Malvaceae',, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniaceae, Theaceae, Umbelliferae. • . *' - : -
  • Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants such as, for example, the Gramineae family, such as rice, maize, wheat or .other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and all grass species.
  • the Gramineae family such as rice, maize, wheat or .other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and all grass species.
  • the invention is applied very particularly preferably to dicotyledonous plant organisms.
  • Preferred dicotyledonous plants are selected in particular from the dicotyledonous crop plants such as, for example,
  • Asteraceae such as sunflower, tagetes or calendula and- others
  • Cruciferae particularly the genus Brassica, very particularly the species napus (oilseed rape) , campestris (beet) , oleracea cv Tastie (cabbage) , oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other cabbages; and the genus Arabidopsis, very particularly the species thaliana, and cress or canola and others,
  • Cucurbitaceae such as melon, pumpkin/squash or zucchini and others ,
  • - Leguminosae particularly the genus Glycine, very particularly the species max (soybean) , soya, and alfalfa, pea, beans or peanut and others, - Rubiaceae, preferably the subclass Lamiidae such as, for example Coffea arabica or Coffea ⁇ iberica (coffee bush) and others ,
  • Solanaceae particularly the genus Lycopersicon, very particularly the species esculentum (tomato) , the genus ⁇ Solanu , very particularly the species tuberosum (potato) and melongena (aubergine) and ' the genus Capsicum, very particularly the genus annuum (pepper) and. tobacco or paprika and others,
  • Sterculiaceae preferably the subclass Dilleniidae such as, for example, Theobro a cacao (cacao bush) and others,
  • Theaceae preferably the subclass Dilleniidae such as, for example, Camellia sinensis or Thea sinensis (tea shrub) and others ,
  • Umbelliferae particularly the genus Daucus (very particularly the species carota (carrot) ) and Apium (very particularly the species graveolens dulce (celery)) and others;
  • Tree species preferably comprise plum, cherry, peach, nectarine, apricot, banana, kiwi, papaya, mango, apple, pear, quince. ;
  • angiosperms such as, for example, Hepaticae (liverworts) and Musci (mosses) ,- pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae, the families of the Rosaceae such as rose, Ericaceae such as rhododendron and azalea, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as ⁇ .
  • bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses)
  • pteridophytes such as ferns, horsetail and clubmosses
  • Iridaceae such as gladioli, iris, freesia and crocus
  • Compositae such as marigold, Geraniaceae such as geranium, Liliaceae such as dracena, Moraceae such as ficus, Araceae such as cheeseplant and many others .
  • Preferred within the scope of the i vention are those plants which are employed as foodstuffs or feeding stuffs .
  • the method of the invention is suitable to introduce modification ' and/or mutations in the chromosomal DNA of a eukaryotic cell or organism, preferably a plant cell or a plant organism.
  • chromosomal DNA or "chromosomal DNA-sequence” is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chro atids, they might be condensed or uncoiled.
  • mutation and “modification” and grammatical equivalents thereof when used in reference to a nucleic acid sequence (such as the chromosomal DNA) are used interchangeably and are to be understood in the broad sense and is intended to include substitution, addition, deletion, inversion or insertion of at least one or more base pairs.
  • a “deletion” is defined as a change in a nucleic acid sequence in which one or more nucleotides is absent.
  • An “insertion” or “addition” is that change in a nucleic acid sequence which has resulted in the addition of one or more nucleotides.
  • insertion is in- tended to comprise single base insertion and/or insertion of additional genes or expression cassettes.
  • a “substitution" results from the replacement of one or more nucleotides by a molecule which is a different molecule from the replaced one or more nucleotides.
  • a nucleic acid may be replaced by a dif- ferent nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine.
  • a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol.
  • Said mutation or modification may affect the coding region as much as the non-coding region (e.g., the 5 '-untranslated, 3'- untranslated, intron or promoter region) of the a gene.
  • Consequences of said mutation may be various and may - for example- cause a decrease or an increase of the amount of mRNA or protein expressed from said gene, or of the function and/or activity of the corresponding gene product . exp > « .
  • the method of the invention is not limited to any particular frequency of modification and/or recombination.
  • the invention's methods result in a frequency of modification in the target nu- cleotide sequence of from 0.1% to 5%. Nonetheless, any frequency (i.e., between 0% and 100%) of modification and/or recombination is contemplated to be within the scope of the present invention.
  • the frequency of modification and/or recombination is dependent on the method used to induce the modification and/or recombination-, the cell type used, the specific gene targeted and' the DNA mutating reagent used, if any. " ⁇
  • the method used to detect the modification and/or recombination may not detect all occurrences of modification and/or recombina'tion.
  • some modification and/or recombination events may be silent, giving no detectable indication that the modification and/or recombination has taken place.
  • the inability to ' ••detect silent modification and/or recombination events gives an artificially low estimate of modification and/or recombination. Because of these reasons, and others, the invention is not limited to any particular modification and/or recombination frequency. In one embodiment, the frequency of modification and/or recombi- nation is between 0.01% and 100%.
  • the frequency of modification and/or recombination is between 0.01% and 50%. In yet another embodiment, the frequency of modification and/or recombination is between 0.1% and 10%. In still yet another embodiment, the frequency of modification and/or recom- bination is between 0.1% and 5%.
  • the present invention is not limited to any degree of precision in the modification and/or recombination of DNA in the cell, it is contemplated that some embodiments of the present invention require higher degrees of precision, depending on the desired result.
  • the specific sequence changes required for gene repair e. g. , particular base changes
  • achievement of higher levels of precision in modification and/or homologous recombination techniques is greater than with prior art methods .
  • Methods for the measurement of DNA modification and/or recombi- nation in the recipient cell or cells may vary depending on the cell type used, the nature of the modification and/or homologous recombination in the cell and the physiological or morphological effect of the DNA- odifying and/or recombination event.
  • the present invention is not limited to any particular method or meth- ods used to determine the DNA modification and/or recombination in the recipient cell or cells.
  • the contemplated methods are well known to those practiced in tfie art.
  • the recombina- tion event is expected to result in a change or changes in a physiological function or a morphological characteristic' in the resulting organism.
  • the expected change or changes can then be" assayed or observed.
  • DNA samples obtained from the organism and changes in gene sequence can be determined by PCR (see, e.g., US 4,683,195, 4,683,202).
  • mutant cell and “modified cell” refer to a cell which contains at least one modification in the cell's genomic sequence.
  • a modification induced by a method of the invention may de demonstrated by PCR analysis, Southern blot analysis, fluorescence in situ hybridization (FISH) , and in situ PCR. These methods may also be used to identify and select a cell comprising said modi- fication of a chromosomal DNA-sequence.
  • FISH fluorescence in situ hybridization
  • the modification may be induced by a DNA-modifying molecule.
  • Said DNA-modifying molecule may , for example, comprise a nucleic acid molecule or a peptide molecule. It may comprise double- and/or single stranded DNA, double- and/or single stranded RNA, or polypeptide molecules.
  • Suitable polypeptide molecules may be for example enzymes with nuclease or recombinase activity.
  • Preferred are eganucleases such like homing-endonucleases (e.g., I-Scel) or recombinase (e.g. Cre or Flp) .
  • TFO triplex-forming oligonucleotides
  • US 5,962,426 oligonucleotides
  • TFO reagents When coupled to a mutagen, TFO reagents are capable of disrupting the sequence of the targeted gene thereby effectively blocking transcription and translation of a functional product.
  • TFOs direct mutagenesis by binding to genomic DNA with sufficient affinity to produce error-prone repair.
  • TFO can act either by delivering a tethered mutagen, such as psoralen or chloram- bucil (Havre et al. (1993) Proc Natl Acad Sci USA 90:7879-7883; Havre et al .
  • TFOs may be tethered to donor duplex DNA (see, e. g., Chan et al. (1999) J Biol Chem 272:11541-11548). TFOs can also act by binding with sufficient affinity to provoke error-prone re'pai " (Wang et al. (1996) Science 271:802-805).
  • TFO Triplex forming oligonucleotide
  • TFO is defined as a sequence of DNA or RNA that is capable of binding in the major grove of a duplex DNA or RNA helix to form a triple helix.
  • a preferred length of the TFO is 200 nucleotides or less, more "*"4 preferably 100 nucleotides or less, yet more preferably from 5 to 50 nucleotides, even more perferably from 10 to 25 nucleo- tides, and most preferably from 15 to 25 nucleotides.
  • TFOs may be generated synthetically, for example, by PCR or by use of a gene synthesizer apparatus. The synthesis of triplex forming oligonucleotides is well known in the art (see US 5,962,426; US 5,874,555).
  • reagents useful for modification of genomic DNA sequences and their synthesis are also well known in the art (e.g., PNAs : US 5,986,053; US 5,539,082; oligomers of cyclic heterocycles: US 5,998,140). It is well know in the art how to induce genomic DNA mutations with PNAs (see, e.g., US 5,641,625).
  • Cook et al. (US 6,025,482) teaches the production of modified oligonucleotide analogs useful in forming triple helix structures with duplex DNA, and also teaches the modification of protein production or function in cells or organisms . Additional methods of inducing modification and/or recombination may be used in conjunction with the present invention. For example methods of recombination utilizing double-crossover events (US 4,873,191; Wagner et al . (1975) Proc Natl Acad Sci USA 72: 3619- 3622; US 4,713,337) .
  • the DNA-modifying molecule may also comprise a DNA-construct.
  • the DNA-construct utilized to introduce modifications and/or mutations into the chromosomal DNA may ' be of various structural configurations. It may have - for example - a linear, linearized or circular structure. It may be double-stranded or single- • « stranded (like e.g., T-DNA at a certain point of its transfer) .
  • the DNA-construct is introduced into the cell in form of a linearized double-stranded DNA or in form of a T-DNA.
  • the DNA-construct may be introduced into the eu- karyotic cell in form of a transposon (comprising the mutation- ' inducing sequence) .
  • eukaryotic cells are selected in which the transposon had integrated close to the target sequence.
  • a transposase e.g., introduced by tran- sient expression or crossing
  • the DNA-construct becomes available' again for homologous recombination.
  • a homology sequence comprised in a DNA-construct is to be understood to comprise sequences of a length of at least 100 base pair, preferably at least . 250 base pair, more, preferably at least 500 base pair, especially preferably at least 1000 base pair, most preferably at.least 2500 base pair.
  • the term "sufficient homology" with respect to a homology sequence comprised in a DNA-construct is to be understood to comprise sequences having a homology to the corresponding target sequence comprised in the chromosomal DNA (e.g., the target sequence A' or B' ) of at least 70 %, preferably at least 80 %, more preferably at least 90 %, especially preferably at least 95 %, more especially preferably at least 99 %, most preferably 100 %, wherein said homology extends over a length of at least 50 base pair, preferably at least 100 base pair, more preferably at least 250 base pair, most preferably at least 500 base pair.
  • the DNA-constructs utilized within the method of this invention may comprise additional nucleic acid sequences.
  • Said sequences may be - for example - localized in different positions with re- spect to the homology sequences.
  • the additional nucleic acid sequences are localized between two homology sequences and may be introduced via homologous recombination into the chromosomal DNA, thereby resembling an insertion mutation of said chromosomal DNA.
  • the additional sequences may also be localized outside of the homology sequences (e.g., at the 5'- or 3 '-end of the DNA-construct).- In cases where the additional sequence resembles a negative selection marker this may allow a distinction of illegitimate insertion events from correct, insertion events mediated by homologous recombination. Corresponding negative markers are described below and suitable methods are well known in the art (WO 99/20780).
  • said sequences are expression cassettes, which may - for example - facilitate expression of selection markers, trait genes, or antisense RNA.
  • said expression cassettes comprise a promoter sequence functional in plant cells opera- tively linked to a nucleic acid sequence which - upon expression - confers an advantageous phenotype to the so transformed plant.
  • the person skilled in the art is aware of numerous sequences which may be utilized in this context, e.g. to increase quality of food and feed or to produce chemicals, fine chemicals ' or pharmaceuticals (e.g., vitamins, oils, carbohydrates) (Dunwel ⁇ " JM (2000) J Exp Bot 51 Spec No : 487-96) .
  • growth, yield, and resistance against abiotic and biotic stress factors may be enhanced.
  • Advantageous properties may be conferred either by overexpressing proteins or by decreasing expression of endogenous proteins by e.g., expressing a corresponding antisense or double-stranded RNA.
  • efficiency of the method of the invention may be further increased by combination with other methods suitable for increasing homologous recombination.
  • Said methods may include for example expression of HR- enhancing proteins (like e.g., RecA; WO 97/08331; Reiss B et al . (1996) Proc Natl Acad Sci USA 93 (7) :3094-3098; Reiss B et al .
  • PARP inhibitors suitable for use within this invention are known to the person skilled in the art and may include for example preferably 3-Aminobenzamid, 8-Hydroxy-2- methylquinazolin-4-on (NU1025) , 1, llb-Dihydro- [2H]benzopyrano[4,3,2-de]isoquinolin-3-on (GPI 6150), 5- ' Aminoisoquinolinon, 3, 4-Dihydro-5- [4- (l-piperidinyl)butoxy] - 1(2H) -isoquinolinon or compounds described in WO 00/26192, WO 00/29384, WO 00/32579, WO 00/64878, WO 00/68206, WO 00/67734,
  • the method may be combined with other methods facilitation homologous recombination and/or selection of the recombinants like e.g., positive/negative selection, excision of illegitimate recombination events or induc- tion of sequence-specific or unspecific DNA double-strand breaks .
  • a nucleic acid molecule whose- expression (transcription and, if appropriate, translation) gener- ates an "anti-CAF” compound is preferably operably linked to at least one genetic control element (for example a promoter) which , ensures expression in an organism, preferably in plants.
  • a genetic control element for example a promoter
  • the expression construct is to be introduced directly into a plant and the "anti-CAF" compound (for example the CAF dsRNA) is to be generated therein in planta, plant-specific genetic control ele- ments (for example promoters) are preferred.
  • the "anti- CAF” compound may also be generate ⁇ iri" other organisms or in vitro and then be introduced into the plant (as described in Examples 6 and 7) .
  • Preferred in this context are all of the prokary- otic or eukaryotic genetic control elements (for example promoters) -which permit the expression. in the organism chosen "in- each case for the preparation. "" "''''
  • Operable linkage is to be understood as meaning, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example an "anti-CAF" compound,) and, if appropriate, further regulatory elements such as, for example, a terminator in such a way that .each of the regulatory elements can fulfill its function when the nucleic aci - sequence is expressed recombinantly, depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules.
  • Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other.
  • the distance be- tween the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
  • Operable linkage, and an expression cassette can be generated by means of customary recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch EF and Sam- brook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al. (1990) In: Plant Molecular Biology Manual.
  • sequences which, ' for example, act as a linker with specific cleavage sites for restriction enzymes, or as a t> signal « peptide, may also be positioned between the two sequences.
  • the insertion of sequences may also lead to the expression of fusion proteins.
  • the expression cassette consisting of a linkage of promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a plant genome, for example by transx rmation.
  • expression cassette also denotes those constructions in which a promoter is positioned behind an endogenous CAF gene, for example by means of homologous recombination, and the decrease according to the invention of an CAF protein is brought ""* "' about by the expression of an antisense CAF RNA.
  • an "anti-CAF" compound for example a nucleic acid sequence encod- ing an CAF dsRNA or an CAF antisense RNA
  • an "anti-CAF" compound can be positioned behind an endogenous promoter in such a way that the same eff'qct is manifested. Both approaches lead to inventive expression cassettes .
  • promoters are known in the art to allow expression in eukaryotic species. Described are promoters suitable for expression in cells of e.g., mammalian, insect, namatode, plant, fungal etc. origin.
  • Suitable eukaryotic promoters may be in particular ubiquitous promoters (HPRT, vimentin, alpha-actin, tubulin, and the like) , promoters of therapeutic genes (of the type including MDR, CFTR, and the like) , tissue-specific promoters (of.
  • promoters of genes for desmin, myosins, creatine kinase, phosphoglycerate kinase or alternatively promoters responding to a stimulus such as promoters responding to the natural hormones (receptor for steroid hormones, receptor for retinoic acid, and the like) or a promoter regulated by antibiotics (tetracyclin, rapamycin, and the like) .
  • they may be promoter sequences derived from the genome of a virus, for example, the promoters of the EIA genes of the adenovirus, MLP genes, or promoters derived from genomes of the viruses CMV, RSV, SV40, and the like.
  • the retroviral LTR (long terminal repeat) is active in most cells (e.g., hematopoietic cells) in vivo and will generally be relied upon for transcription of the inserted sequences and their constitutive expression (Ohashi et al. (1992) Proc. Natl. Acad. Sci. 89:11332; Correll et al. (1992) Blood 80:331).
  • suitable promoters include the human ' cytomegalovirus (CMV) immediate early promoter and the U3 region promoter of the
  • MMSV Moloney Murine Sarcoma Virus
  • SFFV Spleen Focus Forming Virus
  • promoters that may be used to cause expression of the introduced sequence in specific cell types include Granzyme A for expression in T-cells and NK cells, the CD34 promoter for expression in stem and in stem and progenitor cells, the CD8 promoter for expression in cytotoxic T-cells, and the CDllb promoter for expression in mye- loid cells.
  • the promoters may also be inducible or repressible. Inducible promoters include e.g., the tetracycline regulation system and metallothionein promoter (lida et al . (1996) J VIROL. 70:6054-59, 1996; Palmiter (1994) PROC NATL ACAD Sci USA '1 > 91:1219-23).
  • plant-specific promoters are pre- ferred.
  • plant-specific promoter is understood as meaning, in principle, any promoter which is capable of governing the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues or plant cultures.
  • expression can be, for example'' consti- tutive, inducible or development-dependent.
  • Constant promoters refers to those promoters which ensure expression in a large number of, preferably all, tissues over a substantial period of plant development, preferably at all times during plant development (Benfey et al.(1989) EMBO J 8:2195- 2202) .
  • a plant promoter or promoter originating from a plant virus is especially preferably used.
  • the promoter of the CaMV (cauliflower mosaic virus) 35S transcript (Franck et al . • (1980) Cell 21:285-294; Odell et al. (1985) Nature 313:810-812; Shewmaker et al. (1985) Virology 140:281-288; Gardner et al . (1986) Plant Mol Biol 6:221- 228) or the 19S CaMV promoter (US
  • Another suitable constitutive promoter is the Rubisco small subunit (SSU) promoter (US 4,962,028), the leguminB promoter (GenBank Ace. No. X03677) , the promoter of the nopalin synthase from Agrobacterium, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), the ubiquitin 1 promoter (Christensen et al..
  • SSU Rubisco small subunit
  • promoters with specificities for • —4 seeds such as, for example; the phaseolin promoter (US 5,504,200; Bustos MM et al . (1989) Plant Cell 1 (9).: 839-53) , the promoter of the 2S albumin gene (Joseffson LG et al. (1987) J
  • seed-specific promoters are those of the gene encoding high-molecular weight glutenin (HMWG) , gliadin, branching enzyme, ADP glucose pyrophosphatase (AGPase) or starch synthase. Promoters which are furthermore preferred are those which permit a seed-specific expression in monocots such- as maize, barley, wheat, rye, rice and the like.
  • HMWG high-molecular weight glutenin
  • AGPase ADP glucose pyrophosphatase
  • starch synthase starch synthase.
  • the promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzin gene, the prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the casirin gene or the secalin gene) can advantageously be employed.
  • the expression cassettes may also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108), by means of which the expression of the exogenous gene in the plant can be controlled at a particular point in time.
  • a chemically inducible promoter such as, for ,•» example, the PRPl promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186) , a tetracyclin-inducible promoter (Gatz et al.
  • an abscisic acid-inducible promoter EP 0 335 528) or an ethanol-cyclohexanone-inducible ⁇ ro ' moter can likewise be used.
  • the promoter of the glutathione-S transferase isoform II gene (GST-II-27) , which can be activated by exogenously applied safeners such as, for example, N,N-diallyl-2, 2-dichloroacetamide (WO 93/01294 and which is operable in a large number of tissues of both monocots 4 and dicots .
  • constitutive promoters are particularly preferred.
  • promoters may be linked operably to the nucleic acid sequence to be expressed, which promoters make possible the expression in further plant tissues or in other organ- isms, such as, for example, E. coli bacteria.
  • Suitable plant promoters are, in principle, all of the above-described promoters .
  • nucleic acid sequences present in the expression cassettes or vectors according to the invention can be linked operably to further genetic control sequences in addition to a promoter.
  • genetic control sequences is to be understood in the broad sense and refers to all those sequences which have an effect on the materialization or the function of the expression cassette according to the invention. For example, genetic control sequences modify the transcription and translation in prokaryotic or eukaryotic organisms .
  • the expression cassettes according to the invention encompass a promoter functional in plants 5 '-upstream of the nucleic acid sequence in question to be expressed recombinantly, and 3 ' -downstream a terminator sequence as additional genetic control sequence and, if appropriate, further customary regulatory elements, in each case linked operably to the nucleic acid sequence to be expressed recombinantly.
  • Genetic control sequences also encompass further promoters, promoter elements or minimal promoters, all of which can modify the expression-governing properties.
  • tissue-specific expression may additionally ' depend on certain stress- ors, owing to genetic control sequences.
  • Such elements have been described, for example, for water stress, abscisic acid ( (Lam E ... and Chua NH, J Biol Chem 1991; 266(26): 17131 -17135) and heat stress (Schoffl F et al . , Molecular & General Genetics 217(2- 3):246-53, 1989).
  • control sequences are, for example, the Gram-positive promoters amy and SP ⁇ 2 ' and the yeast or fungal promoters ADC1, MFa , AC, P-60, CYCl, ' GAPDH, TEF, rp28, ADH.
  • Genetic control sequences furthermore also encompass the 5'- untranslated regions, introns or noncoding 3 ' -region of gen'e.s, such as, for example, the actin-1 intron, or the Adhl-S introns 1, 2 and 6 (general reference: The Maize . Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994) ) . : .lt has been demonstrated that they may play a significant role in the regulation of gene expression. Thus, it has been demonstrated that 5' -untranslated sequences can enhance the transient expression of heterologous genes . Examples of translation enhancers which may be mentioned are the tobacco mosaic virus 5 ' leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like. Furthermore, they may promote tissue specificity (Rouster J et al . (1998) Plant J 15:435-440).
  • the expression cassette may advantageously comprise one or more of what are known as enhancer sequences, linked operably to the promoter, which make possible an increased recombinant expression of the nucleic acid sequence. Additional advantageous sequences , such as further regulatory elements or terminators , may also be inserted at the 3 ' end of the nucleic acid sequences to be expressed recombinantly. One or more copies of the nucleic acid sequences to be expressed recombinantly may be present in the gene construct.
  • Polyadenylation signals which are suitable as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium t ⁇ mefaciens, in particular gene 3 of the T-DNA (octopin synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984). EMBO'J 3:835 et seq.) or functional equivalents thereof.
  • Examples of terminator sequences which are especially suitable are the OCS (octopin synthase) terminator and the NOS (nopalin synthase) ,• « terminator .
  • the recombinant expression cassette may also com- prise, in particular upstream of the nucleic acid to be ex- pressed, a signal sequence directing the product synthesized in the secretory pathways of the targeTB cell .
  • This signal sequence may be the natural signal sequence of the product, but it may also be any other functional signal sequence, or an artificial signal sequence.
  • the recombinant expression cassette may also comprise a signal sequence directing the synthesized product towards a particular compartment of the cell, such as, for exam- " " * pie, peroxisomes, lysosomes and mitochondria.
  • Control sequences are furthermore to be understood as those which make possible homologous recombination or insertion i ⁇ to the genome of a host organism or which permit removal from the genome.
  • homologous recombination for example the natural promoter of a particular gene may be exchanged' -xfor a promoter functional in plants .
  • Methods such as the cre/lox technology permit a tissue-specific, if appropriate inducible, removal of the expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14 (4) :381-92) .
  • specific flanking sequences (lox sequences) , which later allow removal by means of ere recombinase, are attached to the target gene.
  • An expression cassette and the vectors derived from it may comprise further functional elements .
  • the term functional element is to be understood in the broad sense and refers to all those elements which have an effect on the generation, amplification or function of the expression cassettes, vectors or recombinant organisms according to the invention. The following may be mentioned by way of example, but not by limitation:
  • Selection marker are useful to select and separate successfully transformed or homologous recombined cells.
  • Selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e.g., ' 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or ly ,• « phosate) .
  • a biocidal compound such as a metabolic inhibitor (e.g., ' 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or ly ,• « phosate) .
  • a biocidal compound such as a metabolic inhibitor (e.g., ' 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase conferring resistance to Glyphosate ® (N- (phosphonomethyl) glycine),
  • Glyphosate ® degrading enzymes (Glyphosate ® oxidoreductase; gox)
  • acetolactate syn- thases for example mutated ALS variants with, for example, the S4 and/or Hra mutation
  • NPTII G418- resistance genes coding e.g., for neomycin phosphotransferases
  • aadA gene which confers resistance to the antibiotic spectinomycin the streptomycin phosphotransferase (SPT) gene, which allows resistance to streptomycin and the hygromycin phosphotransferase (HPT) gene, which mediates resistance to hygromycin.
  • SPT streptomycin phosphotransferase
  • HPT hygromycin phosphotransferase
  • Negative selection markers are especially suitable to select organisms with defined deleted sequences comprising said marker (Koprek T et al. (1999) Plant J 19(6): 719-726).
  • Examples for negative selection marker comprise thymidin kinases (TK) , cyto- sine deaminases (Gleave AP et al. (1999) Plant Mol Biol. • 40(2) :223-35; Perera RJ et al. (1993) Plant Mol. Biol 23(4): 793-799; Stougaard J; (1993) Plant J 3:755-761), cytochrom P450 proteins (Koprek et al.
  • Reporter genes encode readily quantifiable proteins and,' 1 via their color or enzyme activity, make possible an assessment of" *4 the transformation efficacy,- the site of expression or the time of expression. Very especially preferred in this context are genes encoding reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(l):29-44) such as the green fluorescent, protein (GFP) (Sheen et al.
  • GFP green fluorescent, protein
  • Origins of replication which ensure amplification of the expression cassettes or vectors according to the invention in, for example, E. coli.
  • Examples which may be mentioned are ORI (origin of DNA replication) , the pBR322 ori or the P15A ori (Sambrook et al. : Molecular Cloning. A Laboratory Manual, 2nded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .
  • a selectable marker which con- fers resistance to a biocide (for example herbicide) , a metabolism inhibitor such as 2-deoxyglucc * se-6-phosphate (WO 98/45456) or an antibiotic to the cells which have successfully undergone recombination.
  • the selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al . (1986) Plant Cell Reports 5:81-84). " ' ' •
  • an expression cassette according to the invention into an organism or cells, tissues, organs., parts or seeds thereof (preferably into plants or plant cells, tissue, organs, parts or seeds) can be effected advantageously using vectors which comprise the expression cassettes .
  • the expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction cleavage site.
  • the plasmid formed is first introduced into E.coli. Correctly transformed E.coli are selected, grown, and the recombinant plasmid is obtained by the methods familiar to the skilled worker. Restriction analysis and sequencing may serve to verify the cloning step.
  • vectors may be plasmids, cosmids, phages, viruses or else agrobacteria.
  • the expression cassette is introduced by means of plasmid vectors .
  • Preferred vectors are those which make possible stable integration of the expression cassette into the host genome.
  • retroviral vector refers to a retrovirus or retroviral particle which is capable of entering a cell and integrating the retroviral genome (as a double-stranded p ' rovi- rus) into the genome of the host cell. Retroviral vectors can be used to transfer genes efficiently into host cells by exploiting the viral infectious process (Kim et al. (1993) Anim Biotechnol 4:53-69; Kim et al.
  • the host range of a retroviral vector i.e., the range of cells that these vec- tors can infect
  • a retroviral vector i.e., the range of cells that these vec- tors can infect
  • an envelope protein from another closely related virus Methods for using retrovi- ruses for the production of transgenic " animals are described (US 6,080,912) and have been established as an efficient and safe route for gene transfer into mammalian cells (Shimotohno & Temin (1981) Cell 26:67-77; Rubenstein et al . (1986) Proc Natl Acad Sci USA 366-68).
  • retroviral vectors in which ' a ⁇ single foreign gene can be inserted include, but are those derived* from plant, murine, avian o primate retroviruses , including but not limited to, Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • murine mammary tumor virus murine mammary tumor virus
  • WO 94/2 ' 9.438 describes the construction of retroviral packaging plasmids and packaging cell lines.
  • the invention furthermore relates to recombinant plant organisms or tissues, organs, parts, cells or propagation material thereof which comprise a recombinant expression cassette ' for "anti-CAF" compound or a recombinant vector encompassing such an expression cassette.
  • Such a recombinant plant organism is generated, for example, by means of transformation or transfection by means of the corresponding proteins or nucleic acids .
  • the generation of a transformed organism requires introducing the DNA in question (for example the expression vector) , RNA or protein into the host cell in question.
  • the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles.
  • the cell can be permeabilized chemically, for example using polyethylene glycol, so that DNA can enter the cell by -diffusion.
  • the DNA can also be introduced by protoplast fusion with other DNA- containing units such as minicells, cells, lysosomes or liposomes .
  • Another suitable method of introducing DNA is electropo- ration, where the cells are permeabilized reversibly by an electrical pulse. Suitable methods have been described (for example by Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991)
  • Suitable meth ⁇ c ⁇ s are especially protoplast transformation by polyethylene-glycol- induced DNA uptake, the biolistic method with the gene gun, what is known as the particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution, and m'icro- injection.
  • trans- formation can also be effected by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • the Agrobacterium-mediated transformation is best suited to dicotyledonous plant cells. The methods are described, for example, by Horsch RB et al. (1985) Science 225: 1229f .
  • the expression cassette is integrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector. If a Ti or Ri plasmid is to be used for the transformation, at least the right border, but in most cases the right and left border, of the Ti or Ri plasmid T-DNA is linked to the expression cassette to be introduced in the form of a flanking region.
  • Binary vectors are preferably used.
  • Binary vectors are capable of replication both in E.coli and in Agrobacterium.
  • they comprise a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border sequence. They can be transferred directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187).
  • the selection marker gene permits the selection of transformed Agrobacteria and is, for example, the nptll gene, which confers resistance to kanamycin.
  • the Agrobacterium which acts as host organism in this case should al- ready contain a plasmid with the vir region. The- latter is re- ' quired for transferring the T-DNA to ' the plant cell.
  • T-DNA for transforming plant cells.
  • the use of T-DNA for transforming plant cells has .• « been studied and described intensively (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287).
  • Various binary vectors are known, some of which are commercially available such as, for example, pBI101.2 or pBINl9 (Clontech Laboratories, Inc. USA). ⁇ " " " " " "
  • Direct transformation techniques are suitable for any organism and cell type.
  • the plasmid used does not need to meet any particular additional requirements in the case of the injection or electroporation of DNA or RNA into plant cells : Simple plasmids such as those of the pUC series can be used. If complete plants are to be regenerated from the transformed cells, it is necessary for an additional selectable marker gene to be located on the plasmid.
  • Stably transformed cells i.e. those which contain the intro- Jerusalem DNA integrated into the DNA of the host cell, can be selected from untransformed cells when a selectable marker is part of the DNA introduced.
  • genes which can act as markers are all those which are capable of conferring resistance to antibiotics or herbicides are given above.
  • Transformed cells which express such marker genes are capable of surviving in the presence of concentrations of a corresponding antibiotic or herbicide which kill an untransformed wild type.
  • the resulting plants can be bred and hybridized in the customary fashion. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary.
  • the construct to be expressed is preferably cloned into a vector which is suitable for the transformation of Agrobacterium tumefaciens, for example pBinl9 (Bevan et al. (1984) Nucl Acids Res 12:8711f).
  • a complete plant can be obtained using methods known to the skilled , worker. For example, callus cultures are used as starting material. The development of shoot and root can be induced in this as yet undifferentiated cell biomass in a known fashion. The shoots obtained can be planted out " and bred.
  • the invention also relates to recombinant organisms transformed with at least one of the nucleic acid sequences according to the invention, expression cassette according to the invention or, vector according to the invention, and to cells, cell cultures, tissues, parts - such as, for example, leaves, roots and the like in the case of plant organisms - or propagation m'aterial derived from such organisms.
  • organism is to be understood in the broad sense and refers to prokaryotic and eukaryotic organisms, preferably bacteria, yeasts, ' fungi, non- human animal organisms and plant organisms . Preferred plant organisms are indicated above.
  • Host or starting organisms which are preferred as recombinant organisms are mainly plants in accordance with the above definition. Included within the scope of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Furthermore included are the mature plants, seed, shoots and seedlings, and parts, propagation material and cultures derived therefrom, for example cell cultures. Mature plants refers to plants at any developmental stage beyond that of the seedling; The term seedling refers to a young immature plant in an early developmental stage. ;
  • the recombinant organisms can be generated with the above- described methods for the transformation or transfection of organisms .
  • the invention furthermore relates to the use of the recombinant organisms according to the invention and of the cells, cell cultures, parts - such as, for example, roots, leaves and the like in the case of recombinant plant organisms - derived from them, and to recombinant propagation material such as seeds or fruits, for the production of foodstuffs or feeding stuffs, pharmaceuticals or fine chemicals.
  • a method for the recombinant production of pharmaceuticals or fine chemicals in host organisms wherein a host organism is transformed with one of the above-described expression cassettes and this expression cassette comprises one or more structural genes which encode the desired fine chemical or catalyze the biosynthesis of the desired fine chemical, the transformed host organism is cultured, and the desired fine chemical is isolated from the culture medium.
  • This method can be applied widely to fine chemicals such as enzymes, vitamins, " ⁇ amino acids, sugars, fatty acids, and natural and synthetic flavorings, aroma substances and colorants.
  • the production of tocopherols and tocotrienols and carotenoids are especially preferred.
  • the transformed host organisms are cultured and the product's, are isolated from the host organisms or the culture medium by methods known to the skilled worker.
  • the production of pharmaceuticals such as, for example, antibodies or vaccines, is .'described by Hood EE, Jilka JM. Curr Opin Biotechnol. 1999 Aug; 10(4) :382- 6; Ma JK, Vine ND. Curr Top Microbiol Immunol. 1999; 236:275-92.
  • Another embodiment of the invention is directed to a process for facilitating plant breeding utilizing a plant organism having a decreased activity or expression of at least one CAF protein.
  • the plant of the invention having said decreased activity or expression of at least one CAF protein exhibits an enhanced rate of meiotic recombination.
  • This property may, for example, be utilized to facilitate breeding programs by e.g., allowing faster segregation of advantageous and disadvantageous traits .
  • this goal can be faster achieved utilizing the plants of the invention.
  • a further alternative . use for the invention's methods is the generation of transgenic cells and transgenic animals which are useful as models for diseases, and for screening therapeutic reagents.
  • a particular modification to a gene in a first animal e. g. , human
  • the invention's methods may be used to introduce the same or similar odifica- tion into the genome of another animal (e. g. , mouse) in-order" to generate a transgenic animal which may be used as a model for the disease in the first animal.
  • Transgenic animals may be generated using several methods which are known in the art, including microinjection, retroviral infection, and implantation of embryonic stem cells. For example, modifications may be introduced into fertilized eggs, cells from pre-implantation embryos such as blastomeres, eight-cell embryos, blastocoele, and midgestation embryos, and into embryonic stem (ES) cells.
  • ES embryonic stem
  • Yet another use of the invention's methods is for targeted re- combination for the purpose of producing gene knockout organisms and/or of replacement of defective genes with non-defective genes . It is well established that the frequencies of existing " ⁇ " 4 protocols for homologous recombination in mammalian cells are quite low. However, using the invention's methods, the frequency can be dramatically increased. This would be useful for the construction of transgenic animals and cell lines as described, above, and also for the correction of existing gene defects at the site of the defect. For example, in one embodiment, it is contemplated that stem cells (e.
  • the methods of the present invention will help ensure the efficiency of the modification and/or recombination phase of the procedure, thereby increasing the likelihood of success.
  • This approach has the advantage of a genetic in correction, rather than the current approach of introducing a separate copy of a gene that integrates at a site other than the natural site, and that is often subject to regulation that is different from the native gene.
  • a further application of the invention's methods is in determining the function of a gene of unknown function. This is of particular interest, given the current concern for characterizing the many new genes described by the Genome Project. For example, if a transgenic animal, which is constructed using the inven- tion ' s methods that result in knockout of a gene of unknown function, were to develop cancer, it could be concluded that the function of the gene was related to the regulation of cellular growth in the tissues in which the cancer originated. Alterna- ' tively, if the animal developed muscular disorders it could be concluded that the novel gene played a role in the normal development and function of muscle tissue. ⁇ ..,
  • the method according to the present invention can be used in gene therapy.
  • gene therapy or “gene transfer” is defined as the insertion of genes into cells for the purpose of medicinal therapy.
  • gene therapy may include expression of a transferred gene, but also the modulation or the blocking of a gene to provide the treatment of a particular pathological condition. -
  • therapeutic gene is understood to mean in particular any gene encoding an RNA or a protein product having a therapeutic effect.
  • the protein product encoded may be a protein, a peptide and the like. This protein product may be homologous in relation to the target cell (that is to say a product which is normally expressed in the target cell when the latter exhibits no pathological condition) .
  • the expression of the transgene makes- it possible, for example, to overcome an inadequate expression in the cell or the expression of an inactive or weakly active protein due to a modification, or makes it possible to overexpress the said protein.
  • the therapeutic gene may also encode a mutant of a cellular protein having increased stability or a modified activity, and the like.
  • the protein product may also be heterologous in relation to the target cell.
  • an expressed protein may, for example, supplement or provide an activity which is deficient in the cell (treatment of enzymatic deficiencies) , or may make it possible to combat a pathological condition, • or to stimulate an immune response for example for the treatment of tumours . ;
  • Illustrative genomic sequences which may be modified using the invention's methods include, but are not limited to, sequences which encode enzymes,- lymphokines (e. g., interleukins , inter- ferons, TNF, etc.) ; growth factors (e. g. , erythropoietin, G- CSF, M-CSF, GM-CSF, etc.) ,- neurotransmitters or their precursors or enzymes responsible for synthesizing them,- trophic factors (e.
  • enzymes e. g., interleukins , inter- ferons, TNF, etc.
  • growth factors e. g. , erythropoietin, G- CSF, M-CSF, GM-CSF, etc.
  • neurotransmitters or their precursors or enzymes responsible for synthesizing them e.
  • BDNF BDNF
  • CNTF NGF, IGF, GMF, aFGF, bFGF, NT3 , NT5 , HARP/pleiotrophin, etc.
  • apolipoproteins e. g. , ApoAI,. Apo-' AIV, ApoE. etc.
  • LPL lipoprotein lipase
  • the tumor- suppressing genes e. g., p53, Rb, RaplA, DCC k-rev, etc.
  • factors involved in blood coagulation e.
  • genomic sequences are those for which a mutant has been associated" with a human disease.
  • genomic sequences are exemplified, but not limited to, the adenosine deaminase (ADA) gene (GenBank Ace. -NO.M13792) associ- ated with adenosine deaminase deficiency with severe combined immune deficiency; ⁇ l-antitrypsin gene (GenBank Ace . -No.Mil465)
  • NP purine nucleoside phosphorylase
  • dystrophin Gene (GenBank Accession Nos. M18533, M17154, and M18026) associated with muscu- lar dystrophy
  • utrophin also called the dystrophin related protein
  • leptin for the treatment of obesity blood pressure regulating factors, such as the enzymes involved in the metabolism of NO, angiotensin, bradykinin, vaso- pressin, ACE, renin, the enzymes encoding the mechanisms for the synthesis or for the relief .
  • blood pressure regulating factors such as the enzymes involved in the metabolism of NO, angiotensin, bradykinin, vaso- pressin, ACE, renin, the enzymes encoding the mechanisms for the synthesis or for the relief .
  • mediators such as histamine, serotonin, catecholamines , neuropeptides, anti- angiogenic factors such as the ligand for Tie-1 and for Tie-2, ' ' " • angiostatin, ATF factor, derivatives of plasminogen, endothelin, thrombospondins 1 and 2, PF-4, ⁇ - or ⁇ -interferon, interleukin-
  • TNF ⁇ urokinase receptor
  • fltl KDR
  • PAI1, PAI2 PAI2
  • TIMPl pro- prolactin fragment
  • factors protecting against apoptosis such ' as the AKT family
  • proteins capable * - ⁇ inducing cell death either active by themselves such as the caspases or of the "pro- drug" type requiring activation by other factors, or proteins activating pro-drugs into an agent causing cell death, such as the herpesvirus thymidine kinase, deaminases, which make'- it possible in particular to envisage anticancer therapies
  • proteins-** involved in intercellular contacts and adhesion VCAM, PECAM, ELAM, ICAM, integrins , cathenins, proteins of the extracellular matrix, proteins involved in the migration of cells proteins of the signal transduction type, of the type including FAK, MEKK, p38 kinase, tyrosines, kinases, serine-threonine
  • transcription factors jun, fos, API, p53, and the proteins of the p53 signalling cascade, cell structure proteins, such as the intermediate filaments (vimentin, desmin, keratins) , dystrophin, the proteins involved in muscle contractility and in controlling muscle contractibility, in particular the proteins involved in calcium metabolism and the flow of calcium in the cells (SERCA) .
  • the ligand or the receptor e.g., FGF-R, VEGR-R etc.
  • HSCs hematopoietic stem cells
  • Diseases- for which gene transfer into HSCs is potentially useful include bone marrow disorders, erythroid cell defects, metabolic disorders and the like.
  • Hematopoietic stem cell gene therapy is beneficial for the treatment of genetic disorders of blood cells such as ⁇ - and ⁇ -thalassemia, sickle cell anemia and hemophilia A and B in which the globin gene or clotting factor gene is de- fective.
  • Another good example is the treatment of severe combined immunodeficiency disease (SCIDS) , in which patients lack the adenosine deaminase (ADA) enzyme.
  • SCIDS severe combined immunodeficiency disease
  • ADA adenosine deaminase
  • Other diseases include chronic granulomatosis where the neutrophils express a defective cytochrome b and Gaucher disease resulting from an abnormal glu- cocerebrosidase gene product in macrophages .
  • variable fragments of single-chain antibody (ScFv) or any other antibody fragment possessing recognition capacities for ''its use in immunotherapy for example for the treatment of infectious diseases, of tumours, of autoimmune diseases such as multiple sclerosis (antiidiotype antibodies) as well as the ScFv's which becomes attached to the pro-inflammatory cytokines such as, for example, ILl and TNF ⁇ for the treatment of rheumatoid arthritis.
  • proteins of interest are, in a nonlimiting manner, soluble receptors such as, for example, the soluble CD4 receptor or the soluble receptor for TNF for anti-HIV therapy, the TNF ⁇ receptor or the ILl soluble receptor for the treatment of rheumatoid arthritis, the soluble receptor for acetylcholine for the treat- ment of myasthenia; substrate peptides or enzyme inhibitors, or peptides which are agonists or antagonists of receptors or of adhesion proteins such as, for example, for the treatment of asthma, thrombosis, restenosis, metastasis or inflammation; artificial, chimeric or truncated proteins.
  • soluble receptors such as, for example, the soluble CD4 receptor or the soluble receptor for TNF for anti-HIV therapy, the TNF ⁇ receptor or the ILl soluble receptor for the treatment of rheumatoid arthritis, the soluble receptor for acetylcholine for the treat- ment of myasthenia
  • insulin in the case of diabetes, growth hormone and calcitonin. It is also possible to mention proteins capable of inducing antitu our immunity or of stimulating the immune response (IL2, GM-CSF, IL12, and the like) . Finally, it is possible to mention the cytokines which reduce the THl response such as IL10, IL4 and 1113.
  • IL10, IL4 and 1113 proteins capable of inducing antitu our immunity or of stimulating the immune response
  • cytokines which reduce the THl response such as IL10, IL4 and 1113.
  • the therapeutic nucleic acid may also be an antisense sequence or gene whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs .
  • Such sequences may, for example, be transcribed in the target cell into RNA complementary to cellular mRNAs and thus block their translation into protein, according (EP-Al 140 308) .
  • the therapeutic genes also comprise the sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP-A1 321 201) .
  • SEQ ID NO : ' 1 Nucleic acid sequence encoding CAF-1 pl50 protein from Arabidopsis thalina
  • SEQ ID NO: 2 Amino acid sequence encoding CAF-1 plSO protein from Arabidopsis thalina
  • SEQ ID NO: 3 Nucleic acid sequence encoding CAF-1 pl50 protein from Homo sapiens
  • SEQ ID NO: 4 Amino acid sequence encoding CAF-1 pl50 protein from Homo sapiens
  • SEQ ID NO: 5 Nucleic acid sequence encoding CAF-1 pl50 protein from Oryza sativa (rice)
  • SEQ ID NO: 6 Amino acid sequence encoding CAF-1 pl50 protein from Oryza sativa (rice)
  • SEQ ID NO: 7 Nucleic acid sequence encoding CAF-1 pl50 protein from Mus musculus (mouse)
  • SEQ ID NO: 8 Amino acid sequence encoding CAF-1 pl50 protein from Mus musculus (mouse)
  • SEQ ID NO: 9 Nucleic acid sequence encoding CAF-1 pl50 protein from Xenopus laevis
  • SEQ ID NO: 10 Amino acid sequence encoding CAF-1 pl50 protein from Xenopus laevis
  • SEQ ID NO: 11 Nucleic acid sequence encoding CAF-1 pl50 protein from Glycine max (soybean) ,- fragment
  • SEQ ID NO: 12 Amino acid sequence encoding CAF-1 pl50 protein from Glycine max (soybean) ,- . fragment
  • SEQ ID NO: 13 Nucleic acid sequence encoding CAF-1 pl50 protein from Brassica napus (canola) ; fragment 14.
  • SEQ ID NO: 14 Amino acid sequence encoding CAF-1 pl50 protein from BrasSica napus (canola) ; fragment
  • SEQ ID NO: 15 Nucleic acid sequence encoding CAF-1 pl50 protein from Brassica napus (canola " )'; > fragment " ⁇
  • SEQ ID NO: 16 Amino acid sequence encoding CAF-1 pl50 protein from Brassica napus (canola) ; fragment
  • SEQ ID NO: 17 Nucleic acid sequence encoding CAF-1 pl50 protein from Glycine max (soybean)';; fragment
  • SEQ ID NO: 18 Amino acid sequence encoding CAF-1 pl50 protein from Glycine max (soybean) ,- fragment
  • SEQ ID NO: 19 Nucleic acid sequence encoding CAF-1 pl50 protein from Oryza sativa (rice) ; fragment
  • SEQ ID NO: 20 Amino acid sequence encoding CAF-1 pl50 protein from Oryza sativa (rice) ,- fragment
  • SEQ ID NO: 21 Nucleic acid sequence encoding PCNA -protein from Arabidopsis thalina
  • SEQ ID NO: 2 Amino acid sequence encoding PCNA protein from Arabidopsis thalina
  • SEQ ID NO: 23 Nucleic acid sequence encoding PCNA protein from Nicotiana tabacum
  • SEQ ID NO: 24 Amino acid sequence encoding PCNA protein from Nicotiana tabacum
  • SEQ ID NO: 25 Nucleic acid sequence encoding PCNA protein from Zea mays (corn)
  • SEQ ID NO: 26 Amino acid sequence encoding PCNA protein from Zea mays (corn) 27.
  • SEQ ID NO: 27 Nucleic acid sequence encoding PCNA protein from Gallus gallus" (chicken) ,- fragment
  • SEQ ID NO: 28 Amino acid sequence encoding PCNA protein from Gallus gallus (chicken) ,- fragment
  • SEQ ID NO: 29 Nucleic acid sequence encoding PCNA protexh from Glycine max (soybean) ,- fragment
  • SEQ ID NO: 30 Amino acid sequence encoding PCNA protein from Glycine max (soybean) ,- fragment
  • SEQ ID NO: 31 Nucleic acid sequence encoding PCNA protein from Homo sapiens; fragment / ' ;.
  • SEQ ID NO: 32 Amino acid sequence encoding PCNA protein from Homo sapiens; fragment ⁇
  • SEQ ID NO: 33 Nucleic acid sequence encoding PCNA protein from Mus musculus (mouse) ,- fragment
  • SEQ ID NO: 34 Amino acid sequence encoding PCNA protein from Mus musculus (mouse) ,- fragment
  • SEQ ID NO: 35 Nucleic acid ' sequence encoding PCNA protein from Rattus norvegicus (rat) ; fragment
  • SEQ ID NO: 36 Amino acid sequence encoding PCNA protein from Rattus norvegicus (rat) ,- fragment
  • SEQ ID NO: 37 Nucleic acid sequence encoding CAF-1 p48 protein from Arabidopsis thalina
  • SEQ ID NO: 38 Amino acid sequence encoding CAF-1 p48 protein from Arabidopsis thalina
  • SEQ ID NO: 39 Nucleic acid sequence encoding CAF-1 p48 protein from Gallus gallus (chicken) .
  • SEQ ID NO: 40 Amino acid sequence encoding CAF-1 p48 protein from Gallus gallus (chicken)
  • SEQ ID NO: 41 Nucleic acid sequence encoding CAF-1 p48 protein from Homo sapiens 42.
  • SEQ ID NO: 42 Amino acid sequence encoding CAF-1 p48 protein from HSitio "sapiens
  • SEQ ID NO: 43 Nucleic acid sequence encoding CAF-1 p48 protein from Danio rerio (zebrafish) ; fragment
  • SEQ ID NO: 42 Amino acid sequence encoding CAF-1 p48 protein from Danio rerio (zebrafish) ; fragment
  • SEQ ID NO: 45 Nucleic acid sequence encoding CAF-1 p48 protein from Glycine max (soybean) ; fragment
  • SEQ ID NO: 46 Amino acid sequence encoding CAF-1 p48 protein from Glycine max (soybean) ,- fragment
  • SEQ ID NO: 47 Nucleic acid sequence encoding CAF-1 p48 protein from Oryza sativa (rice) ,- fragment
  • SEQ ID NO: 48 Amino acid sequence encoding CAF-1 p48 protein from Oryza sativa (rice) ,- fragment
  • SEQ ID NO: 49 Nucleic acid sequence encoding CAF-1 p60 protein from Arabidopsis thalina
  • SEQ ID NO: 50 Amino acid sequence encoding CAF-1 p60 protein from Arabidopsis thalina
  • SEQ ID NO: 51 Nucleic acid sequence encoding CAF-1 p60 protein from Glycine max (soybean)
  • SEQ ID NO: 52 Amino acid sequence encoding CAF-1 p60 protein from Glycine max (soybean)
  • SEQ ID NO: 53 Nucleic acid sequence encoding CAF-1 p60 protein from Homo sapiens
  • SEQ ID NO: 54 Amino acid sequence encoding CAF-1 p60 protein from Homo sapiens
  • SEQ ID NO: 55 Nucleic acid sequence encoding CAF-1 p60 protein from Brassica napus (canola) ,- fragment a*
  • SEQ ID NO : ' 56 Amino acid sequence encoding CAF-1 p60 protein from Brassica napus (canola) ,- fragment
  • SEQ ID NO: 57 Nucleic acid sequence encoding CAF-1 p60 ' ⁇ protein- from Oryza sativa (rice) ,- fragment
  • SEQ ID NO: 58 Amino acid sequence encoding CAF-1 p60 protein from Oryza sativa (rice) ,- fragment
  • SEQ ID NO: 60 Nucleic acid sequence encoding construct "nptll-cassette Kpnl" for monitoring homologous recombination
  • SEQ ID NO: 61 Nucleic acid sequence encoding construct "Als-3'-ncr" for monitoring homologous recombination
  • Fig. la-c Alignment of CAF-1 pl50 proteins from; various species.
  • X. laevis Xenopus laevis
  • CAFA_Human Homo sapiens
  • CAFA_Mouse Mus musculus
  • 0. sativa rice
  • A. thalina Alignment of CAF-1 pl50 proteins from; various species.
  • X. laevis Xenopus laevis
  • CAFA_Human Homo sapiens
  • CAFA_Mouse Mus musculus
  • 0. sativa rice
  • A. thalina Arabidopsis thaliana
  • Fig. 2 Alignment of CAF-1 p60 proteins from various plant species.
  • Zmays Zea mays
  • L.e. Lopersicum esculentum; tomato
  • ' A. thalina Arabidopsis thaliana
  • the consensus sequence indicating regions of homology is given below the individual sequences .
  • Fig. 3a-b Alignment of CAF-1 p60 proteins from various species.
  • the consensus sequence indicating regions of homology is given below the individual sequences .
  • Fig.4a-b Alignment of CAF-1 p48 proteins from various"''species .
  • Glycine max (soybean); Danio rerio (zebrafish); B.napus (Brassica napous; canola); Gallus gallus (chicken); human (Homo sapiens); 0. sativa (rice); A. thalina (Arabidopsis thaliana) .
  • the consensus sequence indicating regions of homology is 'given below the individual sequences .
  • Fig. 5a-b Alignment of PCNA proteins from various species. Zea mays (corn); A. thalina (Arabidopsis thaliana);
  • B.napus Brain napous; canola
  • N. tabacum Naturala tabacum; tabac
  • A. thalina Arabidopsis thaliana; 2nd isoform
  • Glycine max Soybean
  • R.norvegicus Rattus norvegicus; rat
  • PCNA_CHICK Gallus gallus; chicken
  • Mus musculus Muse
  • Fig.6 Scheme of insertion of (S653N) mutation into endogenous AHAS gene (conferring herbicide resistance) by utalization of a homologous recombination gene targeting construct.
  • Fig. 7 Scheme for construction of vector pGEM-Teasy-At .AHAS- R-truncated-At .AHAS-term-nosP-nptll-nosT.
  • Fig. 8 Scheme for construction of vector pGEM-Teasy-At .GTC- 1.
  • oligonucleotides can be synthesized chemically irr* 4 the known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897) .
  • the cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoreses, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial .'cultures, multiplication of phages and sequence analysis of recombinant DNA, are carried out as decribed by Sambrook et al .
  • the plant Arabidopsis thaliana belongs to the higher plants (flowering plants) . This plant is closely related to other plant species from the Cruciferae family such as, for example, Brassica napus, but also to other families of dicotyledonous plants . Owing to the high degree of homology of its DNA sequences or its polypeptide sequences, Arabidopsis thaliana can be employed as model plant for other plant species . ;
  • the plants are grown either on Murashige-Skoog medium supplemented with 0.5 % sucrose (Ogas et al . (1997) Science 277:91-94) or in soil (Focks & Benning (1998) Plant Physiol 118:91-101) .
  • the seeds are first placed on medium or scattered on the .soil ' and then stratified for two days at 4°C. After flowering, the pods are labeled. According to the labels, pods aged 6 to 20 days post-anthesis are then harvested.
  • Nurse r> .
  • Example 2 Plasmids for the transformation of plants
  • Binary vectors such as pBinAR can be used for the transformation of plants (H ⁇ fgen und Willmitzer (190 ' ) Plant Science 66: 221- 230) .
  • the binary vectors can be constructed by ligating the cDNA into T-DNA in sense and antisense orientation. 5' of the cDNA, a plant promoter activates the transcription of the cDNA.
  • a polyadenylation sequence is located 3' of the cDNA.
  • " ' ' • Tissue-specific expression can be achieved using a tissue- ""* 4 specific promoter.
  • seed-specific expression can be achieved by cloning in the napin or the LeB4- or the USP promoter 5' of the cDNA. Any other seed-specific promoter element can also be used.
  • the CaMV 35S promoter can be used , for constitutive expression in the whole plant.
  • Agrobacterium-mediated plant transformation can be carried out for example using the Agrobacterium tumefaciens strains GV3101 (pMP90) (Koncz und Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404 (Clontech) . Standard transformation techniques may be used for the transformation (Deblaere et al.(1984) Nucl Acids Res 13:4777-4788) .
  • Agrobacterium-mediated plant transformation can be effected using standard transformation and regeneration techniques (Gelvin SB, Schilperoort R, Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuch Universitye Signatur: BTll-P ISBN 0-7923-2731-4; Glick BR, ;Thompson JE, Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pp., ISBN 0-8493-5164-2).
  • oilseed rape can be transformed by cotyledon or hypocotyl transformation (Moloney et ' al.(1989) Plant Cell Report 8:238-242; De Block et al.(1989) Plant Physiol 91: 694-701).
  • the use of antibiotics for the selection of agrobacteria and plants depends on the binary vector used for the transformation and the agrobacterial strain.
  • the selection of oilseed rape is usually carried out using kanamycin as selectable plant marker.
  • Agrobacterium-mediated gene transfer into linseed (Linum usitatissimum) can be carried out for ' example using a technique described by Mlynarova et al. (1994) Plant Cell Report 13:282- 285.
  • Soya can be transformed for example using a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, US 5,376,543-, US 5,169,770 (University- of Toledo) . ' ⁇ -
  • transgenic resistant seedlings four-leaf stage
  • the wildtype seedlings are bleached out or dead.
  • Example 6 Studying the expression of a recombinant gene product in a transformed organism
  • the activity of a recombinant gene product in the transformed host organism was measured at the transcription and/or translation level.
  • a suitable method for determining the level of transcription of the gene is to carry out a Northern blot as described hereinbelow (for reference see Ausubel et al . (1988) Current Protocols in Molecular Biology, Wiley: New York, or the above examples section) , where a primer which is designed such . that it binds to the gene of interest is labeled with a detectable label (usually a radiolabel or chemiluminescent label) so that, when the total RNA of a culture of the organism* • is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe, binding and the extent of binding of the probe indicates the presence and the amount of mRNA for this gene.
  • a detectable label usually a radiolabel or chemiluminescent label
  • Cellular total RNA can be ' prepared from cells, tissues or organs ' " using several methods, all of which are known in the art, for example the method Bormann ER et al. (1992) Mol. Microbiol. 6:317-326.
  • RNA hybridization 20 ⁇ g of total RNA or 1 ⁇ g of poly(A) + RNA are separated by means of gel electrophoresis in 1.25% strength agarose gels using formaldehyde and following the method described by Amasino (1986, Anal . ⁇ Biochem. 152, 304) ' ,» transferred to positively charged nylon membranes (Hybond N+, Amersham, Brunswick) by capillary force using 10 x SSC, immobilized by UV light and prehybridized for 3 hours at 68°C using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl, 1 % SDS, 100 mg herring sperm DNA) .
  • hybridization buffer 10% dextran sulfate w/v, 1 M NaCl, 1 % SDS, 100 mg herring sperm DNA
  • the DNA probe is labeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization step, using alpha- 32 P-dCTP (Amersham Pharmacia, Brunswick, Germany) .
  • Hybridization is carried out overnight at 68°C after addition of the labeled DNA probe in the same buffer.
  • the wash steps are carried out twice for 15 minutes using 2 X SSC and twice for 30 minutes using 1 X SSC, 1% SDS, at 68°C.
  • the sealed filters are exposed at -70°C for a period of 1 to 14 days.
  • Example 7 Identification of the CAF gene ..,
  • a population of T-DNA tagged Arabidopsis transformants was generated in the ecotype C24 using Agrobacterium-mediated transformation with vector pAC106 (EMBL nucleotide sequence database ac- cession AJ537513) in strain GV3101pMP90RK (Koncz C & Schell J (1986) Mol Gen Genet 204:383-396) tfsirig vacuum infiltration (Bechtold N et al. (1993) Co ptes Rendus de l 'Academle des Sciences Serie Ill-Sciences de la Vie-Life Sciences 316:194-1199) . This population was screened for visible phenotypes.
  • One plant line ' (named sul ⁇ All) showed obvious growth abnormalities' hen the mutated allele was homozygous .
  • the plants are small, bushy * ' 4 and growth is greatly retarded. Leaves are narrow and rugged. The shoots are fasciated and flowers and siliques abnormal.
  • the gene tagged in sul ⁇ all was isolated by inverse PCR. DNA from homozygous plants was isolated using the CTAB method (Hofmani ⁇ ,, AH et al. (1999) Mol Gen Genet 261:92-99), digested with the restriction endonuclease EcoRI, re-ligated. and purified by ethanol precipitation. 5% of this DNA was used in long template PCR re- action.
  • the reaction conditions were according to the manufactures (formerly Boehringer Mannheim, now Roche) description with the exception that a annealing temperature of 60°C was used.
  • the primers used to amplify T-DNA tag-specific sequences (sense: per sul outl-1, 5 ' -CGGCTCTCATCGAAGAAGGA-3 ' ; anti-sense: per sul out 1-2, 5'-GCTATTGGTCTCGGTGTCGC-3' ) prime inside the sulfonamide resistance gene of pAC106 and read to the outside of the T-DNA.
  • the PCR fragment obtained was digested with Asp718 and Pstl and the fragment cloned in the general purpose cloning vector pUC19.
  • the DNA sequence of the insert showed that the T-DNA has in- serted into chromosome 1 of the Arabidopsis genome.
  • the exact position is at nucleotide 88220 in the DNA sequence of BAG T8F5 (EMBL database accession number AC004512) .
  • This region encodes the fasciata gene and the insertion is in an exon that is located approximately in the middle of the region coding for this gene.
  • the characteristic features of the phenotype of the mutant and the fact that the, insertion is located inside fasciata indicate that sul ⁇ all is allelic to fasciata and that the disruption of this gene causes the mutation.
  • a genomic fragment containing the entire fasciata genomic region including the up- steam region was amplified by long template PCR (formerly Boehringer Mannheim, now Roche) according to the manufactures in- structions using primers fas 2-1 (5 ' -CAAGTTTTATGCCGCCATTA TG-3 ' ) and fas 2-2 (5 ' -CGGTTTTAAC GGCATACGC-3 ' ) and an annealing tem-.., perature of 53°C.
  • the PCR fragment was digested with Pmel and Ncol and cloned into a Smal and Ncol cut derivative of pUC19 containing a modified polylinker.
  • This intermediate was digested with Ncol, the ends filled in with DNA polymerase Klenow frag- ment and the fasciata fragment released by digestion with Sail.'
  • This fragment was cloned into the Binary vector pMOOl (Reiss B et al. (1994) Plant Physiology (Life Science Advances) 13:143- 149) digested with S al and Sail to yield pMl71.
  • pM171 was transformed by electroporation into Agrobacterium GV3101pMP90RK and used to transform heterozygous sul8All plants by vacuum infiltration.
  • Intra-chromosomal homologous recombination is a generally accepted and widely used assay to determine the activity of ho- mologous recombination in plants (Peterhans A et al. (1990) Embo J 9:3437-3445; 1990; Gal S et al . (1991) EMBO J 10:1571-1578; Lebel EG et al. (1993) Proc Nat Acad Sci USA 90:422-426; Puchta H et al. (1994) Experientia 50:277-284; Swoboda P et al . (1994) EMBO J 13:484-489; Puchta H et al .
  • heterozygous sul8All plants and ah independent pACIO6 recombinant line without any visible pheno- type were crossed with the homozygous line 651.E.33 carrying th t e GUS intra-chromosomal recombination reporter gene (Swoboda P et al. (1994) EMBO J 13:484-489).
  • Homologous recombination events in this line are revealed by a reconstitution of an functional GUS gene, the expression of which is visualized by staining of whole tissue or plants.
  • the number- of "blue spots" obtained after staining for GUS activity direc ⁇ ly" reflects the activity of homologous recombination in this system.
  • Gene targeting assays To analyze the efficiency of gene targeting in sul8All defective plants, commonly used gene targeting assay systems are used. Systems that rely on herbicide or antibiotic resistance genes are selected. A variety of assay systems are available in Arabidopsis or are easily adapted to Arabidopsis (Paszkowski J et al . (1988) , EMBO J 7:4021-4026; Halfter U et al . (1992) Mol Gen Genet 231:186-193; Lee KY et al . (1990) Plant Cell 2:415-426; Puchta H et al. (1996) Proc Nat Acad Sci USA 93 : 5055-5060;Reiss B et al . (2000) Proc Nat Acad Sci USA 97:3358-3363; Hanin M et al . (2001) Plant J 28:671-677) . ;
  • Plants containing the targeting reporter genes are generated by transformation or the corresponding endogenous genes converted to repair constructs.
  • the corresponding lines are crossed to heterozygous sul ⁇ All plants, or, when endogenous genes are used, these plants are used directly.
  • Plants obtained from the cross are selected for the presence of the gene targeting reporter genes and the sul ⁇ All mutation using the corresponding select-- able marker genes.
  • Heterozygous plants containing both loci are transformed with the repair construct by vacuum infiltration. In parallel, the same procedure is done with an independent pAC106, recombinant line to determine the basic gene targeting frequencies in the system.
  • the seeds obtained from infiltrated plants are selected on the substances designed to reveal gene targeting events and the gene targeting events in positive lines confirmed by PCR and Southern blots as described (Reiss B et al. (2000) Proc Nat Acad Sci USA 97 : M et al. (2001) Plant J 28:671-677).
  • a direct comparison of the targeting frequencies between control and sul8All lines shows the stimulatory effect of CAFl deficiency.
  • Other transformation methods will be used in addition to Agrobacterium-mediated transformation. Homozygous sul ⁇ All plants are difficult to transform by vacuum infiltratio'n since they barely set flowers and seeds. However, homozygous material can easily be grown in large quantities in tissue cul- ture.
  • vectors carrying the same selectable markers are transformed in protoplasts, roots, shoots, leaves, and calli of heterozygous and homozygous sul ⁇ All and C24 wild-type plants by electroporation and particle bombardment.
  • the number of calli actively growing on selective media directly indicates the efficiency of transformation. For experiments a significantly increased efficacy of transformation can be observed.
  • Example 10 Determination of the gene targeting frequency at the A. thaliana AcetoTSctate synthase (Als) -locus.
  • the Acetolactate synthase catalyzes the initial step in the formation of the branched-chain amino acids in plants .
  • the ALS-protein is the target of several classes of herbicides' e.g. , imidazolinones (RAPTOR 8 imazamox; PURSUIT ® imazethapyr) . From the literature mutated and imidazolinone resistant versions of the Als gene are known (Lee KY et al . (1988) EMBO J 7 (5) :1241-1248) .
  • GTC gene targeting construct based on an Als allele encoding a herbicide insensitive ALS variant.
  • This gene targeting construct (GTC) consists of three different elements:
  • Homologous recombination events between the Als gene on the chromosome and the recombination substrate result in the replacement of the c-terminal part of the Als wildtype gene with the truncated mutated Als gene and the insertion of the nptll expression-cassette between the coding sequence of the Als gene and- the adjacent 3' non coding region (Fig. 6).
  • the use of the described gene targeting construct as recombination substrate leads to a dominant phenotype (resistance against Imidazolinone herbicides and Kanamycin) uppn successful gene targeting.
  • the transgenic plants acquire resistance against Kanamycin but they are still sensitive against Imidazolinone herbicides.
  • the resistance against Kanamycin is used to determine the overall transformation frequency covering the illegitimate and the homologous recombination evenfs". ' "This frequency has to be determined in order to calculate the HR frequency at the targeted locus .
  • Example 8.2 Cloning and mutagenesis of a truncated A. ' 'thaliana ecotype Columbia Als wildtype allele --*
  • the genomic sequence of the Als locus from A. thaliana ecotype Columbia was cloned by the use if the PCR technology (SEQ ID NO: 59) .
  • SEQ ID NO: 59 The genomic sequence of the Als locus from A. thaliana ecotype Columbia was cloned by the use if the PCR technology (SEQ ID NO: 59) .
  • sequence specific primers Using sequence specific primers, a truncated version of the Als gene lacking 724 basepairs at the 5' end of the coding region was amplified from genomic DNA isolated from A . thaliana ecotype Columbia.
  • An additional Sail restriction • '_. site was introduced by a mismatch in the At .Als-trune-5 ' primer leading to an A ⁇ C transversion at position 26 of the PCR fragment.
  • PCR temperature profile 120 seconds 94°C; 30 cycles of 30 seconds 94°C, 60 seconds 54°C, and 90 seconds 68°C; subsequently 600 seconds 72°C (post-elongation) and afterwards storage at 4°C until further use.
  • the amplified fragment was cloned into the PCR cloning vector pGEM ® -Teasy (Promega) resulting in the vector pGEM-Teasy- At.AHAS-truncated-At.AHAS-term.
  • the identity of the amplified genomic fragment was confirmed by sequence analysis (Seq ID NO: 59) .
  • sequence analysis Seq ID NO: 59
  • a mutation at position 1249 was introduced into the wildtype allele.
  • the mutagenesis was performed according to the manual of the in vitro site directed mutagenesis kit QuikChange ® XL from Stratagene. The introduction of the mutation was confirmed by sequence analysis. The resulting plasmid (pGEM-Teasy-A. tAHAS-R- truncated-At .AHAS-term) was used r fbr "the subsequent production of the gene-targeting construct.
  • Example ⁇ .3 Cloning of the neomycinphosphotransferase (nptll) expression cassette.
  • a PCR fragment representing the nptll expression cassette consisting of the nos promoter, the nptll gene, and the nos termi- nator was amplified from the vector pSUN-1 (PCT/EP/0107359) .
  • the 5 ' primer (nptll 5 ' primer: 5 ' -ggtaccCACTGATAGTTTAAACTGAAG ⁇ -3 ' ) and the 3' primer (nptll 3' primer: 5 ' -ggtaccgatatc GCCCGATCTAG- TAACATAGATG-3 ' ) used for this amplification were flanked by restriction sites for the endonucleases Kpnl, and Kpnl followed by EcoRV, respectively.
  • the amplified fragment was cloned into the PCR vector pGEM-Teasy (pGEM-Teasy-nptll-cassette-Kpnl) .
  • the identity of the amplified fragment was confirmed by sequence analysis (Seq ID NO: 60) .
  • PCR was performed in a 50 ⁇ l Reaction- mix containing:
  • PCR temperature profile 120 seconds 94°C; 30 cycles of 30 seconds 94°C, 90 seconds 60°C, and 90 sec- onds 6 ⁇ °C; subsequently 600 seconds 72°C (post-elongation) and afterwards storage at 4°C until further use.
  • Example ⁇ .4 Cloning of 3' non coding region of the Als gene from A. thaliana ecotyp'e ' Columbia
  • the genomic sequence of the 3 ' non coding region of the Als lo- cus from A. thaliana ecotype Columbia was cloned by the use if the -PCR technology (SEQ ID NO 61) . Using sequence "''specific primers the DNA was amplified from genomic DNA isolated fr ⁇ m A . thaliana ecotype Columbia-.
  • the 5' primer (At.Als3 'ncr-5 ' : 5'- gatatcGTATACATGATCGGTGGTAC-3 ' ) and the 3 'primer (At .A2s-3 'ncr- 3': 5 ' -gatatcccgggACATTCCCATATTCGAACG-3 ' ) used for this amplification were flanked by specific DNA sequences representing the target sequence for the restriction endonucleases EcoRV and EcoRV followed by Smal, respectively.
  • the amplified fragment was cloned into ' the PCR vector pGEM-Teasy (pGEM-Teasy-At .A-Is-3 'ncr) . The identity of the amplified fragment was confirmed by sequence analysis. PCR was performed in a 50 ⁇ l reactionmix containing:
  • PCR temperature profile 120 seconds 94°C; 30 cycles of 30 seconds 94°C, 60 seconds 59°C, and 90 seconds 6 ⁇ °C; subsequently 600 seconds 72°C (post-elongation) and afterwards storage at 4°C until further use.
  • Example 8.5 Production of the A . thaliana Als gene targeting construct.
  • the Als gene targeting construct was produced by three sequen- tial cloning steps: In the first step the neomycinphosphotrans- ferase (nptll) expression cassette was cloned into the vector pGEM-Teasy-At.AHAS-R-truncated-At.AHAS-term by the use of the restriction sites Kpnl.
  • the resulting vector pGEM-Teasy-At .AHAS- R-truncated-At .AHAS-term-nosP-nptll-nosT was digested with EcoRV and the DNA fragment At.Als-3 'ncr (isolated from pGEM-Teasy- At.Als-3 'ncr via restriction digest with EcoRV) was ligated into this linearized vector leading to the production, of the vector pGEM-Teasy-At.AHAS-R-trunc-At.AHAS-term-nosP-nptll-nosT- At-.Als3'ncr (pGEM-Teasy-At .GTC-1) .

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Abstract

La présente invention concerne des méthodes de production d'un organisme eucaryote recombinant, de préférence une plante recombinante, qui consiste à réduire la capacité d'accumulation de la chromatine dudit organisme. La méthode de l'invention peut être utilisée pour introduire des séquences dans l'ADN chromosomique d'une cellule eucaryote, soit par insertion illégitime, soit, de préférence, par recombinaison homologue. Cette méthode donne lieu à une efficacité de transformation accrue et/ou une fréquence accrue de la recombinaison homologue. L'invention concerne également des constructions d'expression appropriées pouvant exprimer au moins une partie d'une séquences d'acide nucléique codant pour une protéine du facteur d'accumulation de la chromatine (p. ex., sous forme d'ARN antisens ou bicaténaire) pouvant réduire de manière appropriée l'expression ou la fonctionnalité d'au moins une protéine du facteur d'accumulation de la chromatine chez une plante ou des cellules végétales. L'invention concerne en outre des cellules végétales ou des plantes recombinantes présentant une capacité d'accumulation de la chromatine réduite, de préférence une expression ou une fonctionnalité réduite d'au moins un facteur d'accumulation de la chromatine; et l'utilisation desdites plantes recombinantes pour la production d'aliments, de produits de nourrissage, de semences, de produits pharmaceutiques ou de produits chimiques de laboratoire.
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Publication number Priority date Publication date Assignee Title
CN112048487A (zh) * 2020-08-10 2020-12-08 山东大学 淀粉酶合成的调控蛋白及其编码基因与应用
CN112048487B (zh) * 2020-08-10 2023-07-04 山东大学 淀粉酶合成的调控蛋白及其编码基因与应用

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